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 [...].

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.

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.

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.

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.

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.

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.

Enhanced recovery of Salmonella Typhimurium DT104 from exposure to stress at low temperature.

Salmonella enterica serovar Typhimurium (S. Typhimurium) remains an important cause of food-borne infection in the developed world. In order to establish infection within a host, Salmonella must survive and recover from a range of environmental stresses. S. Typhimurium strain SL1344 is among the most extensively studied pathogenic Salmonella strains, while S. Typhimurium phage type DT104 is an important type that has been associated with pandemic spread and a high number of food-borne disease outbreaks over the last two decades. In this study, we have compared the abilities of these two S. Typhimurium types to recover from stress exposures commonly encountered in food production, including 685 mM NaCl, pH 3.8, low temperature (6 °C) and combinations thereof. Following removal from prolonged (8 days) stress, DT104 cultures that had been exposed to low temperature, with or without additional stress, resumed exponential growth more rapidly than SL1344 cultures exposed to the same conditions. SL1344 showed higher levels of filamentation than DT104 in response to NaCl exposure at low temperature. Further, SL1344 incurred higher levels of membrane damage in response to elevated NaCl and pH 3.8 at both temperatures compared with DT104. However, both strains recovered normal cell division and membrane integrity within 6 h when all stresses were removed. Expression of the Salmonella pathogenicity island 1 gene prgH, the first gene in the prg/org operon, was monitored using a chromosomal reporter in which gfp(+) expression was driven by the prgH promoter. Recovery of prgH expression was comparable for SL1344 and DT104 exposed to stress at 22 °C. However, DT104 cultures exposed to pH 3.8 or combined NaCl and low-pH stress at low temperature resumed prgH expression more rapidly than SL1344. Both strains recovered maximal levels of prgH expression after 6 h recovery from all stresses and, interestingly, maximal levels of prgH expression were significantly higher in SL1344, consistent with prgH expression in late-exponential, non-stressed SL1344 and DT104 cultures. Together, these data show that S. Typhimurium is capable of rapid recovery from environmental and food-related stresses, and give insight into the enhanced ability of DT104 compared with SL1344 to adapt to such stresses, which may contribute to the success of this globally disseminated pathogenic phage type.

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.

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.

Regulation of Salmonella-induced membrane ruffling by SipA differs in strains lacking other effectors.

The Salmonella pathogenicity island 1 (SPI-1) type three secretion system (TTSS) is essential for Salmonella invasion of host cells through its triggering of actin-dependent membrane ruffles. The SPI-1 effectors SipA, SopE, SopE2 and SopB all have actin regulating activities and contribute to invasion. The precise role of actin regulation by SipA in Salmonella invasion remains controversial since divergent data have been presented regarding the relationship between SipA and membrane ruffling. We hypothesized that the contribution of SipA to membrane ruffling and invasion might vary between Salmonella strains. We compared the effects of SipA deletion on Salmonella enterica serovar Typhimurium (S. Typhimurium) strains that possess or lack SopE. Loss of SipA reduced invasion in the early stages of infection by SopE(+) and SopE(-) strains but the number of membrane ruffles elicited was unaffected. Salmonella strains lacking both SipA and SopE induced ruffles with very different morphology from those induced by wild-type strains or ones lacking single effectors, including the presence of highly dynamic finger-like protrusions and numerous filopodia. A similar phenotype was found for sipA(-)sopE(-), sipA(-)sopE2(-) and sipA(-)sopB(-) mutants. Thus, SipA plays a more prominent role in induction of invasion-competent membrane ruffles by Salmonella lacking a full complement of SPI-1 effectors.

The mechanisms used by enteropathogenic Escherichia coli to control filopodia dynamics.

Enteropathogenic Escherichia coli (EPEC) subverts actin dynamics in eukaryotic cells by injecting effector proteins via a type III secretion system. First, WxxxE effector Map triggers transient formation of filopodia. Then, following recovery from the filopodial signals, EPEC triggers robust actin polymerization via a signalling complex comprising Tir and the adaptor proteins Nck. In this paper we show that Map triggers filopodia formation by activating Cdc42; expression of dominant-negative Cdc42 or knock-down of Cdc42 by siRNA impaired filopodia formation. In addition, Map binds PDZ1 of NHERF1. We show that Map-NHERF1 interaction is needed for filopodia stabilization in a process involving ezrin and the RhoA/ROCK cascade; expression of dominant-negative ezrin and RhoA or siRNA knock-down of RhoA lead to rapid elimination of filopodia. Moreover, we show that formation of the Tir-Nck signalling complex leads to filopodia withdrawal. Recovery from the filopodial signals requires phosphorylation of a Tir tyrosine (Y474) residue and actin polymerization pathway as both infection of cells with EPEC expressing TirY474S or infection of Nck knockout cells with wild-type EPEC resulted in persistence of filopodia. These results show that EPEC effectors modulate actin dynamics by temporal subverting the Rho GTPases and other actin polymerization pathways for the benefit of the adherent pathogen.

LuxS-based quorum sensing does not affect the ability of Salmonella enterica serovar Typhimurium to express the SPI-1 type 3 secretion system, induce membrane ruffles, or invade epithelial cells.

Bacterial species can communicate by producing and sensing small autoinducer molecules by a process known as quorum sensing. Salmonella enterica produces autoinducer 2 (AI-2) via the luxS synthase gene, which is used by some bacterial pathogens to coordinate virulence gene expression with population density. We investigated whether the luxS gene might affect the ability of Salmonella enterica serovar Typhimurium to invade epithelial cells. No differences were found between the wild-type strain of S. Typhimurium, SL1344, and its isogenic luxS mutant with respect to the number and morphology of the membrane ruffles induced or their ability to invade epithelial cells. The dynamics of the ruffling process were also similar in the wild-type strain (SL1344) and the luxS mutant. Furthermore, comparing the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion profiles of wild-type SL1344 and the luxS mutant by Western blotting and measuring the expression of a single-copy green fluorescent protein fusion to the prgH (an essential SPI-1 gene) promoter indicated that SPI-1 expression and activity are similar in the wild-type SL1344 and luxS mutant. Genetic deletion of luxS did not alter the virulence of S. Typhimurium in the mouse model, and therefore, it appears that luxS does not play a significant role in regulating invasion of Salmonella in vitro or in vivo.

The effect of oxide overlayers on secondary electron dopant mapping.

The International Technology Roadmap for Semiconductors ranks dopant profiling as one of the most difficult challenges for analysis of semiconductors. Dopant mapping in the scanning electron microscope (SEM) has the potential to provide a solution. This technique has not yet found widespread application, however, mainly due to the lack of a comprehensive theoretical model, uncertain quantification, and its inability to differentiate doping levels in n-type silicon. Although a Monte Carlo model was recently published that closely matched experimental data obtained in p-doped silicon to data obtained from the theoretical model, a large discrepancy between experimental data obtained for n-type silicon was found. Here we present a Monte Carlo model that provides close matches between experimental and calculated data in both n- and p-type silicon, paving the way for a widespread application of SEM dopant contrast.

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.

Applications of cell imaging 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 both humans and warm-blooded animals. Understanding the mechanisms by which Salmonella induce 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 III secretion system (T3SS). Once these Salmonella-induced responses have resulted in bacterial invasion, proteins translocated by a second T3SS initiate further modulation of cellular activities to enable survival and replication of the invading pathogen. These processes contribute to Salmonella entry into the host and the clinical symptoms of gastrointestinal and systemic infection. 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 microscopical methods to examine Salmonella infection in models ranging from whole animal to isolated cells and simple eukaryotic organisms. For example, electron microscopy and confocal microscopy can reveal the juxtaposition of Salmonella, its products, and cellular components at high resolution. Simple light microscopy (LM) can also be used to investigate the interaction of bacteria with host cells and has advantages for live cell imaging, which enables 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 several LM techniques routinely used in our own research.

Use of fluorescence imaging to investigate the structure and function of intestinal M cells.

Fluorescence imaging technology can be applied to many aspects of cell biology ranging from the analysis of specific markers in cells and tissues to the biological actions and distribution of fluorescent proteins or particles in living cells. In this review, we examine the role of fluorescence imaging, in conjunction with other microscopical techniques, to study sites of uptake of material across the gastrointestinal epithelium. We will focus primarily on intestinal M cells, specialised antigen-sampling cells in the epithelium of the gut-associated lymphoid tissue (GALT), including Peyer's patches. In addition to their importance as sites for uptake of inert material, and hence their potential as a route of delivery of vaccines, etc., M cells are also a major site of infection by a range of microbial pathogens. The application of new fluorescence imaging technologies has expanded our knowledge on the structure, development and function of these fascinating cells.