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MICROBIOLOGY: ON BACTERIAL INJECTION OF EFFECTOR PROTEINS

The following points are made by S. Normark et al (Science 2005 307:1211):

1) Many Gram-negative bacteria interact with human, animal, or plant hosts by injecting effector proteins into the cytosol of host cells through the so-called type III secretion system (injectisome) [1]. The symptoms of infectious diseases such as bubonic plague, shigellosis, salmonellosis, typhoid fever, and infantile diarrhea, largely depend on the repertoire of bacterial proteins injected by the type III secretion system, and what they do once inside eukaryotic host cells. The injectisome is remarkably well conserved among different bacterial pathogens [2]. The multicomponent base structure spans the bacterial periplasm and is associated with both the inner and outer membranes of the bacterium.[3] The filamentous needle, composed of a single protein, projects beyond the bacterial surface. For bacterial effector proteins to be translocated into host cells, the tip of the needle must make contact with the eukaryotic host cell membrane.

2) The enteric pathogen Shigella flexneri possesses a type III secretion system that enables invasion of the gut epithelial cells of mammalian hosts. Invasion provokes an extensive inflammatory reaction in the gut mucosa, a hallmark of shigellosis (bacterial dysentery) [4]. Shigella species cause more than 1 million deaths per year from dysentery and diarrhea To survive host inflammatory processes such as increased production of antibacterial peptides, Shigella is equipped with a lipolysaccharide structure in its outer membrane that contains protective repeat units of an O-antigen polysaccharide. This O-antigen polymer extends beyond the bacterial cell surface and potentially could sterically impede the type III secretory apparatus. However, as West et al [5], Shigella has developed an way of ensuring that its injectisome needle remains operational without compromising the ability of the O-antigen polymer to protect against host inflammatory mediators.

3) West et al (5) used signature-tagged mutagenesis to identify colonization-defective S. flexneri mutants in a rabbit model of shigellosis. From these attenuated mutants, they identified two genes residing on the gtrA, gtrB, gtrV operon of a resident bacteriophage. This operon directs the addition of a glucose residue to each O-antigen repeat unit; this glucosylation step imparts serotype specificity to different strains of S. flexneri. Substantially attenuated virulence was observed in glucosylation-defective gtr mutants of different serotypes; virulence could be restored by introducing a serotype-specific gtr operon. The gtr mutants could still produce lipopolysaccharide with the correct number of O-antigen repeats, and could withstand noxious conditions in the gut such as bile salts, complement-mediated lysis, and gut-specific antibacterial peptides. However, compared with wild-type S. flexneri carrying glucosylated O-antigen, the gtr mutants were considerably less invasive and were less able to provoke an inflammatory response, suggesting a defect in the type III secretion system. An IpaB monoclonal antibody, recognizing the tip of the needle complex, revealed a much lower exposure of the needle at the bacterial cell surface in the glucosylation-defective gtr mutants.

4) How does bacteriophage-mediated glucosylation of the O-antigen affect exposure of the type III secretion needle? West et al [5] used electron microscopy and three-dimensional molecular modeling to show that O-antigen glucosylation results in a conformational change from a linear extended form of the repeating O-antigen polymer to a more compact structure. This modification allows surface exposure of the protruding needle (which is roughly 60 nm long), without compromising the protective role of the O-antigen polymer.

References (abridged):

1. R. Rosqvist, K. E. Magnusson, H. Wolf-Watz, EMBO J. 13, 964 (1994)

2. A. P. Tampakaki et al., Cell Microbiol. 6, 805 (2004)

3. T. C. Marlovits et al., Science 306, [1040] (2004)

4. P. Cossart, P. J. Sansonetti, Science 304, [242] (2004)

5. N. P. West et al., Science 307, 1313 (2005)

Science http://www.sciencemag.org

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MICROBIOLOGY: ON BACTERIAL SECRETED VIRULENCE PROTEINS

The following points are made by B.K. Coombes et al (Current Biology 2004 14:R856):

1) Bacterial pathogens have evolved highly sophisticated mechanisms to adapt to their host environment in order to promote successful replication and dissemination. Bacterial subsistence in the context of the host environment involves a complex interplay between bacterial survival strategies and the ensuing host response to infection, which in many cases serves to limit the replication of the microorganism and prevent its dissemination within the body and to new hosts.

2) The host response to infection begins with the identification of a foreign invader through the use of innate immune surveillance systems, which alert the body to an infection and induce a suite of inflammatory molecules that ultimately shape the adaptive arm of the immune response. The importance of this central defense mechanism -- called "innate immunity" -- is exemplified by its presence in organisms across all kingdoms, including insects, nematodes, plants, and in more complex mammals where it serves as the front-line defense against microorganisms before the adaptive immune response is initiated [1,2].

3) The ability to detect, respond to, and destroy invading microbes is a central tenet for survival, and thus forms a formidable barrier that successful pathogens must overcome [3,4]. This is especially true of infecting microbes that rapidly replicate and induce disease symptoms that overwhelm the innate defenses of the host and transmit to a new host before the adaptive immune response can be activated, but is also necessary for "stealth" pathogens that replicate more slowly and cause persistent infections by interfering with both innate and adaptive immune pathways [5]. The innate arm of the immune response is vigorous and intuitive, responding to molecular signatures characteristic of many pathogens, as opposed to unique antigens that ultimately shape the exquisite specificity and clonal nature of adaptive immunity.

4) In order to persist within and exploit their chosen host in the face of a robust innate immune response, bacterial pathogens often secrete virulence factors (or effectors) into host cells or into the surrounding space that in some way modify host biology to the benefit of the pathogen. The host cell targets of secreted bacterial proteins are diverse, including cytoskeletal components, host cell receptors, an array of signaling molecules, and other host proteins compartmentalized in various organelles. Intense research into this area is beginning to elucidate the repertoire of effector targets, although the mechanism of their action is often more elusive. The modification of host cell pathways allows incoming microbes to replicate in a desired niche in the body, quite often inside host cells, or sometimes on the surface of a host cell. This is the first step in bacterial colonization and pathogenic microbes must still overcome a large hurdle in order to be successful -- surviving, replicating and disseminating in the face of host immunity.

5) There are several strategies that microbes employ to circumvent and overcome innate responses. Many bacterial pathogens have evolved sophisticated secretion systems to inject bacterial molecules into animal and plant cells. These type III and type IV secretion systems are complex organelles that span the Gram-negative envelope, and form a conduit to deliver proteins (and, in the case of type IV systems, DNA) into host cells. The general components of type III and type IV secretion systems are usually conserved, but the particular translocated effectors that enter into host cells are often unique to particular pathogens. The repertoire of effectors dictates the choice of host, how the pathogen behaves inside that host, and ultimately the disease.

6) Many, but certainly not all, effectors are key virulence factors, that is, they are required to cause disease. These effectors target host cellular processes including those linked to innate responses, disrupting or altering these pathways as part of the disease process. The cellular targets are numerous, although those of choice are often central regulators of essential processes, such as cytoskeletal dynamics and vesicular trafficking. Although currently the number of examples is limited, it is possible that for most central cellular processes there is at least one microbial pathogen that targets it.

References (abridged):

1. Janeway, C.A.Jr. and Medzhitov, R. (2002). Innate immune recognition. Annu. Rev. Immunol. 20, 197-216

2. Medzhitov, R. and Janeway, C.A.Jr. (1998). An ancient system of host defense. Curr. Opin. Immunol. 10, 12-15

3. Rosenberger, C.M. and Finlay, B.B. (2003). Phagocyte sabotage: disruption of macrophage signalling by bacterial pathogens. Nat. Rev. Mol. Cell Biol. 4, 385-396

4. Hornef, M.W., Wick, M.J., Rhen, M. and Normark, S. (2002). Bacterial strategies for overcoming host innate and adaptive immune responses. Nat. Immunol. 3, 1033-1040

5. Merrell, D.S. and Falkow, S. (2004). Frontal and stealth attack strategies in microbial pathogenesis. Nature 430, 250-256

Current Biology http://www.current-biology.com

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PUNCTURE OF EUKARYOTIC CELLS BY BACTERIAL NEEDLES

Notes by ScienceWeek:

Biologists classify living systems into two distinct types, cells without an internal membrane-bound genome-carrying nucleus (prokaryotes), and cells (or organisms composed of such cells) that do have an internal membrane-bound genome-carrying nucleus (eukaryotes). Prokaryotes consist of two types of bacteria, the archaebacteria and the eubacteria, with continuing controversy concerning the early evolution of these groups.

The prefix "eu-" means "true". In the first instance above, "eukaryotes" means "true kernel" i.e., "true nucleus". In the second instance above, "eubacteria" means "true bacteria", which is more confusing than useful.

Bacteria are indeed "primitive" organisms. But consider the vital statistics: Each bacterium contain 4000 or 5000 distinctly different proteins, each protein type a separate functional entity, and most of these proteins are precisely and dynamically arranged in space in a system that completely replicates itself approximately every 20 minutes. There are no trivialities when confronting such an apparatus, and the nontriviality is amplified when considering that minute quantities of some of these organisms are capable of killing other organisms a million times their size.

Most bacteria can be classified into two types, depending on the chemistry of their outer coat, which chemistry determines whether a bacterium will admit certain dyes into the interior. The classification, according to the differential staining technique, is "gram-negative" vs. "gram-positive", named after the bacteriologist H.C. Gram (1853-1938). Gram-positive bacteria take up a crystal violet stain and turn purple, while gram-negative bacteria exclude the crystal violet and counterstain instead with stains such as safranin, eosin red, or brilliant green. As might be expected, since the technique differentiates the outer coats of bacteria, some antibiotics are effective against one type and not the other type, and vice versa.

In general, gram-positive bacteria have a structure consisting of a cytoplasmic core, a plasma membrane, and a rigid external capsule. Gram-negative bacteria, however, have two plasma membranes between the inner cytoplasmic core and the external capsule: the two plasma membranes are separated by a "periplasmic space" packed with various enzymes.

Depending on the species, bacteria have various types of extensions protruding through the external capsule and into the ambient medium. One or more "flagella" may be present, long filamentous and flexible structures whose mechanical motions provide the bacterium with motility. Two types of rigid extensions are common, "pili" (Latin for "hairs") and "fimbriae" (Latin for "fringes") (singular: pilus, fimbrium), both much shorter than flagella, and fimbriae much shorter than pili. These structures are 7 nanometers in diameter, and much thinner than flagellae (which are 25 nanometers in diameter). Both pili and fimbriae are believed to be involved in the adhesion of pathogenic bacteria to the surfaces of host cells. The report below concerns a new and third type of external extension --penetrating "needles" that come into existence only upon contact with a host cell.

The bacterial species Yersinia enterocolitica is a pathogen related to Yersinia pestis, the cause of plague. Yersinia enterocolitica is found in the intestinal tract of a variety of animals, the bacterium usually causing disease in these animals. Like Y. pestis, this pathogen is transmissible from animals to humans, in whom it can produce a variety of clinical syndromes. Both Y. pestis and Y. enterocolitica are gram-negative rods.

Among the distinguishing characteristics of prokaryotes is their ability to exchange small packets of genetic information. In many cases, this genetic information is carried on "plasmids", small and specialized extrachromosomal genetic elements that are capable of replication within at least one prokaryotic cell line. In some cases, plasmids may be transferred from one bacterial cell to another via a "sex pilus" that serves as a conduit for the transfer of genetic material -- thus effecting the transmission of specialized genetic information (including virulence characteristics) through a bacterial population.

The following points are made by E. Hoiczyk and G. Blobel (Proc. Natl. Acad. Sci. 2001 98:4669):

1) A number of pathogenic gram-negative bacteria are able to secrete specific proteins across three membranes: the inner and outer gram-negative bacterial membrane and the eukaryotic plasma membrane. In the pathogen Y. enterocolitica, the primary structure of the secreted proteins, as well as the components of the secretion machinery, both plasmid-encoded, is known. But the mechanism of protein translocation from bacterium to host cell is largely unknown.

2) The authors report that Y. enterocolitica polymerizes a 6-kilodalton protein of the secretion machinery into needles that are able to puncture the eukaryotic plasma membrane. These needles apparently form a conduit for the transport of specific proteins from the bacterial to the eukaryotic cytoplasm, where they exert their cytotoxic action. Electron microscopy demonstrates the needles are 60 to 80 nanometers long, 6 to 7 nanometers wide, and contain a hollow center of approximately 2 nanometers in diameter. The authors state their data indicate that it is the polymerization of the 6-kilodalton protein into these needles that provides the force to perforate the eukaryotic plasma membrane. The authors conclude: "Surprisingly, the basic principle of such an injection mechanism is widespread among life forms and is found in organisms as diverse as insects, snails, and fish."

Proc. Nat. Acad. Sci. http://www.pnas.org

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