Introduction to Gene Transfer and Evolution

Gene transfer can be categorized into horizontal (lateral) and vertical pathways. Horizontal gene transfer conveys genetic material between strains or species, while vertical gene transfer involves clonal reproduction and internal evolutionary forces that may involve mutations, recombination and rearrangement within the genome 1, 2 . In order for gene transfer to be successful, the genes must be stably integrated into the genome and fixed within the population 1-3 . The recent expansion in the...

Microbial RNA Extraction RNA Stabilization

There are a number of obstacles that must be overcome to successfully profile microbial mRNA from intracellular models of infection. The most fundamental of these is the extraction of a representative mRNA population. The mRNA content of a microbe will change as differences are sensed in the environment in which the microbe finds itself, and this response is likely to affect the RNA profile within minutes, if not seconds 12 . The extraction process must therefore attempt to limit potential...

Infection of Drosophila Phagocytes

Drosophila melanogaster cells such as primary macrophage-like phagocytes (hemocytes, differentiated plasmatocytes) and phagocytic cell lines can also be used to analyze the host-pathogen interaction. A major advantage of using Drosophila cells is the huge number of mutants that are defective in different aspects of the immune response and the relative ease with which single gene activities can be repressed by inhibitory RNA. The D. melanogaster-derived cell line S2, which was originally...

References

1 Fadok, V.A. and Henson, P.M. (1998) Apoptosis getting rid of the bodies. Curr Biol, 8, R693-R695. 2 Aderem, A. and Underhill, D.M. (1999) Mechanisms of phagocytosis in macrophages. Annu Rev Immunol, 3 Rabinovitch, M. (1995) Professional and non-professional phagocytes an introduction. Trends Cell Biol, 5, 85-87. 4 Underhill, D.M. and Ozinsky, A. (2002) Phagocytosis of microbes complexity in action. Annu Rev Immunol, 20, 825-852. 5 Pluddemann, A., Mukhopadhyay, S. and Gordon, S. (2006) The...

Foreword

Over the last thirty years, the combined use of bacterial genetics, molecular and cell biology, and more recently genomics, have illuminated our understanding of the virulence of the major human and veterinary pathogens. Among those, intracellular bacterial pathogens have played a dominant role in this endeavor because they recapitulate most of the existing steps of microbe-host interaction. The progress made has been impressive, not only conceptually, but also technically. Imaging, for...

The Book

If none of the microorganisms associated with man did any damage, and none was notably beneficial, they would be interesting but relatively unimportant objects. Cedric A. Mims in The Pathogenesis of Infectious Disease, 1988. Since Elie Metchnikoffs first observations of phagocytosed bacteria, intracellular pathogens have attracted the attention of microbiologists and attempts have been made to describe their biology in a systematic and comparative way 70 . In this book, specialists studying the...

Phagocytosis An Overview

The uptake of large (> 0.5 mm) particles by cells, a process termed phagocytosis, has been recognized as an important mechanism for the internalization and subsequent destruction of pathogens by specialized cells of the immune system. In addition, phagocytosis plays a key role in the nutrition of unicellular organisms such as the free-living amoeba Dictyostelium, and in the clearance of apoptotic cells that is central to tissue remodeling and development. Macrophages and neutrophils have been...

Content Mixing versus Membrane Mixing Assays

Fusion of two organelles includes two distinct events mixing of membranes and mixing of contents, both of which can be used to detect phagosome-endosome fusion in cell-free systems. So far, the only example of a membrane mixing assay is that introduced by Peyron et al. 15 . These workers labeled lysosome membranes with the fluorescent phospholipid analogs lissamine rhodamine phosphatidyletha-nolamine (N-Rh-PE) and benzoxadiazole phosphatidylethanolamine (NBD-PE) in concentrations sufficiently...

Intraphagosome Ion Concentrations

Measurement of intraphagosome ion concentrations comes with problems similar to those encountered in real-time detection of enzymatic phagosome activities. While quite a few very useful probes have been developed in recent years 29, 30 , most of them cannot be firmly attached to microbial surfaces as they are. Such surface labeling would allow the quantification of intraphagosome ion concentrations while avoiding overlaying signals from other compartments as the signal is strictly...

Everybody Has A Home of Their Own The Phagosome

Most facultative and obligate intracellular microorganisms have specialized in multiplication within a membrane-bound compartment in their host cells. Their phagosomes either (i) do not mature into phagolysosomes, but share some characteristics with endocytic compartments, or (ii) are compartments that have no obvious connection with the endocytic continuum at all, or (iii) mature into phagolysosomes, but maturation is slowed by the pathogens to allow them to adapt to this environment. To add...

Methods Used to Study Phagosome Biogenesis

Phagosome biogenesis is a temporally and spatially ordered process which, in many aspects, parallels endosome maturation (see Chapters 3 and 5). Interest in microbial phagosome biogenesis has increased considerably in the past decade as cell biologists have recognized the usefulness of pathogen-host cell systems in the study of vesicle trafficking and the cytoskeleton and as microbiologists started to incorporate the more complex biology of eukaryotic cells into their views of infection. While...

Use of Fluorescence Activated Cell Sorting FACS in Phagosome Research

FACS, although a well-established technique in immunological and hematological research, has only been used in a few phagosome studies. There are a number of reports on cell homogenates or partially purified phagosomes which were immu-nostained and analyzed by FACS for the presence and quantities of the respective markers on phagosomes 58-61 . In a modified protocol, pH was quantified in latex bead-containing phagosomes using a similar labeling technology as described above 62 and actin...

Introduction The Evolution of Intracellular Life Forms and their Niches

As species are produced and exterminated by slowly acting and still existing causes, and not by miraculous acts of creation and by catastrophes and as the most important of all causes of organic change is one which is almost independent of altered and perhaps suddenly altered physical conditions, namely, the mutual relation of organism to organism, - the improvement of one being entailing the improvement or extermination of others. Charles Darwin

Complement Receptor Mediated Phagocytosis

The CR3 complement receptor is the second best characterized phagocytic receptor. It is an aMp2 integrin and it has been largely recognized to bind particles opsonized with the complement component C3bi. Several studies have suggested that the mechanisms of ingestion elicited by the FcgR and CR3 receptors are different. Electron microcopy studies showed that while during FcgR-mediated uptake thin, actin-enriched pseudopods extend around the target particle, complement-opsonized particles...

Concluding Remarks

This chapter presents an abbreviated and simplified summary of the events that occur during phagocytosis. While the host intends these responses to clear invading microorganisms, all too often these manage to evade or overcome the microbicidal responses and manage to survive and in some cases to proliferate within the phagocytic cell itself. Subsequent chapters in this volume will describe the individual, ingenious strategies that microbes have evolved to avoid and in some instances even co-opt...

In Vitro Studies

Latex bead phagosomes can be purified by flotation in a sucrose gradient under different conditions, for example, different time-points. This allows the study ofthe influence of proteins and lipids present on the phagosomal membrane in actin binding or assembly (Figure 8.3). In addition, LBPs can be incubated with different Figure 8.3 In vitro assays to monitor actin in latex bead phagosomes (LBPs). (a) Scheme showing the purification of LBPs from macrophages for biochemical analysis. Briefly,...

Microscopic versus Biochemical Assays

Two alternatives to observe organelle fusion in a cell-free system are microscopical or biochemical detection. For microscopic assays each of the fusion partners is tagged with a microscopically detectable label and organelle fusion presents itself as colocalization of both labels. All published microscopic in vitro phagosome-endosome fusion assays are based on electron microscopy. Endocytic compartments are labeled with electron-dense gold nanoparticles via endocytic uptake, while phagosomes...

Acknowledgments

This chapter was supported in part by Public Health Service grants from the National Institutes of Health, R01AI059647 and R01 AI039499 (to D. Dean), and the Founder Region Fellowship (to N. Somboonna). 32 2 Limited Genomes and Gene Transfer in the Evolution of Intracellular Parasitism and Symbiosis References 1 Lawrence, J.G. (1999) Curr Opin Microbiol, 2, 519-523. 2 Koonin, E.V. and Galperin, M.Y. (1997) Curr Opin Genet Dev, 7, 757-763. 3 Koonin, E.V., Makarova, K.S. and Aravind, L. (2001)...

FcgRMediated Phagocytosis

The binding of IgG-opsonized targets to their cognate receptors, FcgR, leads to their rapid internalization by a process driven by a remarkable actin-dependent rearrangement of the phagocyte plasma membrane. Actin remodeling is a biphasic event, with initial polymerization during pseudopod extension followed by rapid depo-lymerization during or immediately after sealing of the nascent vacuole 76 . The signals triggering this response will be described next. Particle binding induces clustering...

Gene Transfer in Intracellular Symbionts

Unlike intracellular bacterial parasites, intracellular symbionts have a mutual or commensal relationship with their hosts 6 . Mutualism describes a relationship where both species receive a fitness gain, and commensalism is where one gains but the other is not significantly harmed 86 . However, living together allows symbiotic species to genetically coevolve in a way that can benefit both 87 . Thus, symbiosis is regularly referred to as mutualism 88, 89 . Given that many organisms share...

Dictyostelium a Surrogate Host for Legionella Mycobacterium and Other Pathogens

Legionella Route Infection

Different bacterial species are taken up with different efficiencies and not all phagocytosed bacteria are killed by D. discoideum. In recent years D. discoideum has been established as a host model for several pathogens, including Pseudomonas aeruginosa 24, 25 , Mycobacterium avium, M. marinum 26, 27 , Vibrio cholerae 28 , Klebsiella pneumoniae 29 and Legionella pneumophila 30, 31 . The research areas presently pursued include the use of Dictyostelium wild-type cells as screening system for...

Come On In Get Killed Death in the Shredder

Phagosome Definition

Phagocytosis is defined as the ingestion of a particle by a cell see Chapter 3 . Phagocytosis in vertebrates is typically carried out by professional phagocytic cells, that is, polymorphonuclear cells particularly neutrophils , dendritic cells, monocytes and macrophages, but is not unique to them. Most ingested particles are apoptotic cells, microorganisms and denatured protein complexes larger than some 0.4 mm in diameter. Their surface molecules are recognized by a variety of phagocyte...

Gene Transfer in Intracellular Bacterial Parasites

Trafficking for intracellular bacteria can be categorized into intraphagosomal and intracytoplasmic pathways, in which different relationships with the host cell compartment may involve different modes ofgene transfer and different types ofacquired genes. Intraphagosomal pathogens include Afipia, Brucella, Burkholderia, Chlamydia, Coxiella, Ehrlichia, Francisella, Legionella, Mycobacterium, Rhodococcus, Nocardia and Salmonella, while intracytoplasmic pathogens include Listeria, Rickettsia and...

Burkholderia pseudomallei

Stevens 22.1 Burkholderia pseudomallei and Melioidosis 393 22.2 An Overview of the Intracellular Life of B. pseudomallei 394 22.3 Genome Structure and Diversity of B. pseudomallei 395 22.4 Cell Contact and Adherence 396 22.9 Intracellular Replication 405 22.10 Immunology of B. pseudomallei-Host Cell Interactions 406 22.11 Concluding Remarks 407 References 408

Dictyostelium a Professional Phagocyte at the Border of Multicellularity

Dictyostelium cells are so-called social amoebae which live in the forest soil, feed on bacteria by phagocytosis and proliferate by binary fission 6 . As the food supply diminishes, the haploid cells stop growing and enter the social phase oftheir life cycle by assembling into multicellular aggregates. In a complex developmental process, approximately 105 cells form a migrating slug and finally a fruiting body, consisting of dead stalk cells and spores. The phagocytic potential of Dictyostelium...

The Whole Animal Model Drosophila melanogaster

D. melanogaster mount both cellular and humoral immune responses to pathogens. The innate immune response of the fruit fly D. melanogaster is characterized by a cellular immune response which depends on circulating phagocytic cells, a melanization response which produces reactive oxygen species at sites of infection, and the production ofantimicrobial peptides in the fat body. D. melanogaster lacks an antibody-mediated adaptive immune response 52 , but can react to different kinds of infections...

Phagosome Maturation

After a foreign particle is taken up, the recently formed phagosome undergoes a process of maturation. In essence, phagosomes mature through a series of fusion and fission events with the endocytic pathway. The result of this process is the gradual modification of the phagosomal membrane and contents, ultimately yielding a hybrid organelle known as the phagolysosome. The content of the phagolysosome is recognized by its very low pH, the presence of hydrolytic enzymes and bactericidal peptides...

Look Through the Microscope of Evolution

Mixotricha Paradoxa Diagram

Now, here, you see, it takes all the running you can do, just to keep in the same place. The Red Queen in Lewis Carroll's Alice in Wonderland. The Origin of Species by the Means of Natural Selection, 1859 According to Darwin, life may have started in some warm little pond. Rather than in warm ponds, it is believed today that primitive Bacteria and Archaea arose in the vicinity of hot vents in the ancient oceans some 4 billion years ago and microbes still rule the earth today. It was only after...

Legionelladirected Phagosome Biogenesis

Phagocytosis in D. discoideum and macrophages is known to be regulated by a heterotrimeric G protein-linked signal transduction Table 4.1 . By using pharmacological inhibitors and Dictyostelium mutants it has been demonstrated that L. pneumophila uptake occurs by conventional phagocytosis which requires hetero-trimeric G proteins and the PLC pathway. Moreover, these experiments revealed that cytoplasmic calcium levels, the cytoskeleton proteins coronin, villidin and a-actinin filamin as well as...

9702 52 M J 14 Ms

1 Hayward, R.E., Derisi, J.L., Alfadhli, S., Kaslow, D.C. etal. 2000 Mol Microbiol, 35, 6-14. 2 Monahan, I.M., Betts, J., Banerjee, D.K. and Butcher, P.D. 2001 Microbiology, 147, 459-471. 3 Molestina, R.E., Klein, J.B., Miller, R.D., Pierce, W.H. et al. 2002 Infect Immun, 70, 2976-2981. 4 Florens, L., Washburn, M.P., Raine, J.D., Anthony, R.M. et al. 2002 Nature, 419, 520-526. 5 Heuer, D., Kneip, C., Maurer, A.P. and Meyer, T.F. 2007 Int J Med Microbiol, 297, 569-576. 6 Eyles, J.E., Unal, B.,...

Quantification of Phagosome pH

The first probe developed to explore the intraphagosome environment was a pH sensor. Taking advantage of an assay developed to measure endosome pH 8, 9 , particles were surface labeled with a pH-sensitive fluor, usually fluorescein, whose emission at 520 nm quantifies the intracellular pH due to its exquisite sensitivity when excited at 490 nm 10, 11 . Fluorescein's maximum utility is in the pH range from 5.0 to 7.5, but pH-sensitive fluors with different pH ranges e.g., Oregon Green are also...

Short History of Theories and Discoveries

Clamydia Microscope

The complex mutual relationship between intracellular microbes and their host cells is a challenging field of research and requires the perspective of evolution biology. The individual host-microbe interactions covered in this book all raise the following questions how do microbes enter, survive and proliferate in, and how do they exit host cells And how can intracellular niches be characterized and what are the benefits of intracellular life for the microbes and its consequences for the host...

Particle Induced Impact on Phagosome Isolation

The phagocytic cell encounters a large diversity of particles that vary in size, shape, stiffness and surface properties. For example, red blood cells are round, deformable and between 6 and 40 mm in size bacteria are rather stiff, round, spiral- or rod-shaped, 1 mm in size but can often be found in grapes or strings, like staphylococci or streptococci respectively. Dead cells and most bacteria are negatively charged while mycobacteria are hydrophobic 48 . Accordingly, particles are recognized...

Quantification of Lysosome Components in Phagosomes

Fusion of phagosomes with lysosomes is central to the destruction and killing of ingested materials and microorganisms see Chapter 10 . Phagosome-lysosome fusion is likely to occur in two ways, both of which may act on the same phagosome i complete fusion of preexisting lysosomes with the maturing phagosome and ii transient fusion events between lysosomes and phagosomes, during which only some lysosome contents are transferred into phagosomes and possibly vice versa . The latter has been termed...

Evolution of Intracellular Parasitism

Many microorganisms of diverse phylogenetic beginnings have adapted to intracellular life, each in its own unique way, and sometimes remarkable resemblances in behaviour among intracellular parasites are best ascribed to convergence in similar intracellular habits rather than to divergence from a common origin. Finally it should be remembered that adaptation to intracellular life, although by no means rare, is not easy. After all, most parasites still live extracellularly. James W. Moulder 1985...