Isolation of bacteria-containing phagosomes by magnetic selection

<p>Abstract</p> <p>Background</p> <p>There is a growing awareness of the importance of intracellular events in determining the outcome of infectious disease. To improve the understanding of such events, like phagosome maturation, we set out to develop a versatile technique for phagosome isolation that is rapid and widely applicable to different pathogens.</p> <p>Results</p> <p>We developed two different protocols to isolate phagosomes containing dead or live bacteria modified with small magnetic particles, in conjunction with a synchronized phagocytosis protocol and nitrogen cavitation. For dead bacteria, we performed analysis of the phagosome samples by microscopy and immunoblot, and demonstrated the appearance of maturation markers on isolated phagosomes.</p> <p>Conclusion</p> <p>We have presented detailed protocols for phagosome isolation, which can be adapted for use with different cell types and prey. The versatility and simplicity of the approach allow better control of phagosome isolation, the parameters of which are critical in studies of host-bacteria interaction and phagosome maturation.</p

Phagocytosis of Streptococcus pyogenes by all-trans retinoic acid-differentiated HL-60 cells: roles of azurophilic granules and NADPH oxidase.

BACKGROUND: New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule-phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules. CONCLUSIONS/SIGNIFICANCE: The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion

Phagocytosis by neutrophils - studies on phagosome dynamics and membrane traffic modulation by Streptococcus pyogenes

Neutrophils are our most numerous and deadly white blood cells and without them we would succumb quickly to infections by pathogens. The main mechanism that the neutrophils employ for our protection is phagocytosis, where they eat and enclose their target inside a membrane-bound organelle, the phagosome. Neutrophil phagosomes are highly dynamic entities, and a large amount of antimicrobial substances are released to their interior within seconds of formation. In most cases this will kill the engulfed microbe, but there are exceptions. Streptococcus pyogenes is one of our most common pathogens and is responsible for a wide range of diseases. Recently it has been demonstrated that these bacteria are able to survive phagocytosis by neutrophils. This doctoral thesis is about explaining those mechanisms. What is described in this thesis is the development of new methods for studying phagosome biogenesis and maturation, including creating magnetic bacteria to isolate phagosomes and advanced microscopy for live measurements of phagosomal pH. The employment of cell lines and their differentiation into neutrophils is shown to be a useful research tool. Using these methods, novel findings regarding mechanisms for fusion between neutrophil granules and phagosomes are demonstrated. It is shown that S. pyogenes interfere with the intracellular membrane traffic of neutrophils, leading to a decreased delivery of antimicrobial substances and impaired acidification of S. pyogenes-containing phagosomes, helping them to survive phagocytosis

Preface

This detailed volume presents a diverse set of methodological approaches designed to improve our understanding of bacterial infections from a wide range of bacterial species. Beginning with biofilms and subcellular compartments, the book explores transcriptional analysis, methods for studying plasmid dynamics, and tools for phylogenetic analysis of bacterial genomes, as well as bacterial effector proteins interfering with host systems, host response analysis, and in vivo and in vitro infection models. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Bacterial Pathogenesis: Methods and Protocols, Second Edition is a vital resource for researchers in the area of infection biology, as well as but not limited to, those working in the fields of microbiology, immunology, structural biology, molecular biology, genetics, imaging, and computational study

Automated Image-Based Quantification of Neutrophil Extracellular Traps Using NETQUANT

Neutrophil extracellular traps (NETs) are web-like antimicrobial structures consisting of DNA and granule derived antimicrobial proteins. Immunofluorescence microscopy and image-based quantification methods remain important tools to quantitate neutrophil extracellular trap formation. However, there are key limitations to the immunofluorescence-based methods that are currently available for quantifying NETs. Manual methods of image-based NET quantification are often subjective, prone to error and tedious for users, especially non-experienced users. Also, presently available software options for quantification are either semi-automatic or require training prior to operation. Here, we demonstrate the implementation of an automated immunofluorescence-based image quantification method to evaluate NET formation called NETQUANT. The software is easy to use and has a user-friendly graphical user interface (GUI). It considers biologically relevant parameters such as an increase in the surface area and DNA:NET marker protein ratio, and nuclear deformation to define NET formation. Furthermore, this tool is built as a freely available app, and allows for single-cell resolution quantification and analysis

Measuring Antibody Orientation at the Bacterial Surface

Many bacteria have the ability to interact with antibodies as a means to circumvent the immune response. This includes binding to the Fc portion of antibodies, effectively reversing the antibody orientation and thus decreasing the Fc-mediated immune signaling. Since antibody orientation at the bacterial surface has been shown to be important in human disease, it is valuable to be able to assess how antibodies are interacting with bacterial pathogens. Here, we describe a method to measure the proportion of human IgG that are bound via their Fc or Fabs to a bacterial surface. This is achieved by treating antibody-coated bacteria with the bacterial enzyme IdeS - which will cleave IgG into Fc and Fab fragments - and subsequently detect remaining fragments with fluorescent Fabs. The method is easy and fast, and the principle is most likely also applicable to other systems where distinguishing between antibody Fc and Fab binding is important

Computer Vision-Based Image Analysis of Bacteria

Microscopy is an essential tool for studying bacteria, but is today mostly used in a qualitative or possibly semi-quantitative manner often involving time-consuming manual analysis. It also makes it difficult to assess the importance of individual bacterial phenotypes, especially when there are only subtle differences in features such as shape, size, or signal intensity, which is typically very difficult for the human eye to discern. With computer vision-based image analysis - where computer algorithms interpret image data - it is possible to achieve an objective and reproducible quantification of images in an automated fashion. Besides being a much more efficient and consistent way to analyze images, this can also reveal important information that was previously hard to extract with traditional methods. Here, we present basic concepts of automated image processing, segmentation and analysis that can be relatively easy implemented for use with bacterial research

Bacterial Pathogenesis : Methods and Protocols

This volume discusses various methods and protocols used for the experimentation of a wide range of bacterial species, such as Streptococcus pyogenes, Staphylococcus aureus, Streptococcus pneumonia, Listeria monocytogenes, and Mycobacterium marinum. Bacterial Pathogens: Methods and Protocols is divided into 6 parts: Part 1 describes different approaches to identifying and characterizing bacterial effector molecules; Part 2 deals with structural biology of bacterial pathogenesis and how to overcome folding and stability problems with recombinantly expressed proteins; Part 3 details methodology that identifies bacteria in complex communities and how genomes of bacterial pathogens have evolved; Part 4 reflects on the rapid development of advanced imaging techniques that address questions about molecular properties of individual live bacteria, ultrastructure of surfaces, and subcellular localization of bacterial proteins; Part 5 describes methods from in vitro and in vivo modeling of bacterial infections; and Part 6 explores how bacterial pathogens are the true experts of the immune system. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.Cutting-edge and comprehensive, Bacterial Pathogens: Methods and Protocols is a valuable resource for anyone who is interested in this fascinating and evolving field