C3stau, a new member of the family of C3-like ADP-ribosyltransferases.

C3-like ADP-ribosyltransferases, which are produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus, are exoenzymes lacking a translocation unit. These enzymes specifically inactivate Rho GTPases in host target cells. Recently, a novel C3-like transferase from S. aureus with new properties was identified, raising questions regarding its function. As Rho GTPases are master regulators of several eukaryotic signal processes and S. aureus can invade eukaryotic cells, C3 might play a role as a virulence factor.     

CNF and DNT

The actin cytoskeleton of mammalian cells is involved in many processes that affect the growth and colonization of bacteria, such as migration of immune cells, phagocytosis by macrophages, secretion of cytokines, maintenance of epithelial barrier functions and others. With respect to these functions, it is not surprising that many bacterial protein toxins, which are important virulence factors and causative agents of human and/or animal diseases, target the actin cytoskeleton of the host. Some toxins target actin directly, such as the C2 toxin produced by Clostridium botulinum. Moreover, bacterial toxins target the cytoskeleton indirectly by modifying actin regulators such as the low-molecular-mass guanosine triphosphate (GTP)-binding proteins of the Rho family. Remarkably, toxins affect these GTPases in a bidirectional manner. Some toxins inhibit and some activate the GTPases. Here we review the Rho-activating toxins CNF1 and CNF2 (cytotoxic necrotizing factors) from Escherichia coli, the Yersinia CNF_Y and the dermonecrotic toxin (DNT) from Bordetella species. We describe and compare their uptake into mammalian cells, mode of action, structure–function relationship, substrate specificity and role in diseases.     

Homogeneity and Molecular Weight of Toxin of Clostridium botulinum Type B

Gerwing et al. described the isolation and purification from culture filtrates of the toxin of Clostridium botulinum type B and characterized it as a homogeneous protein of less than 10,000 molecular weight. Analysis by various methods of samples of this toxin obtained from Gerwing et al., and preparations produced by their methods in our laboratories, furnished convincing evidence that neither her preparation nor ours was homogeneous. The molecular weight of the toxic component isolated from either of the preparations was 100,000 or greater and resembled, in a number of respects, the alpha component isolated by us from the crystalline toxin of C. botulinum type A.     

Signaling mechanisms that regulate actin-based motility processes in the nervous system

Actin-based motility is critical for nervous system development. Both the migration of neurons and the extension of neurites require organized actin polymerization to push the cell membrane forward. Numerous extracellular stimulants of motility and axon guidance cues regulate actin-based motility through the rho GTPases (rho, rac, and cdc42). The rho GTPases reorganize the actin cytoskeleton, leading to stress fiber, filopodium, or lamellipodium formation. The activity of the rho GTPases is regulated by a variety of proteins that either stimulate GTP uptake (activation) or hydrolysis (inactivation). These proteins potentially link extracellular signals to the activation state of rho GTPases. Effectors downstream of the rho GTPases that directly influence actin polymerization have been identified and are involved in neurite development. The Arp2/3 complex nucleates the formation of new actin branches that extend the membrane forward. Ena/VASP proteins can cause the formation of longer actin filaments, characteristic of growth cone actin morphology, by preventing the capping of barbed ends. Actin-depolymerizing factor (ADF)/cofilin depolymerizes and severs actin branches in older parts of the actin meshwork, freeing monomers to be re-incorporated into actively growing filaments. The signaling mechanisms by which extracellular cues that guide axons to their targets lead to direct effects on actin filament dynamics are becoming better understood.     

Crystal structure of the C3bot-RalA complex reveals a novel type of action of a bacterial exoenzyme

C3 exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix-loop-helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix alpha3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.     

Inactivation of Clostridium botulinum toxin by ruminal microbes from cattle and sheep.

Toxin from Clostridium botulinum type C was rapidly inactivated during incubation in vitro with ruminal contents from either a cow or a sheep. Fractions of ruminal contents from which cells had been removed by high-speed centrifugation did not inactivate toxin. Inactivation was associated with fractions containing bacteria, whereas fractions containing protozoa and relatively few bacteria were much less active. This activity may help explain the relatively greater tolerance by ruminants to oral doses of botulinum toxin than to toxin administered by other routes. The results are also pertinent to assays for botulinum toxin from gastrointestinal samples obtained postmortem.     

New "hogs" in Hedgehog transport and signal reception

A recent paper in Cell (Yao et al., 2006) and two papers in Developmental Cell (Tenzen et al., 2006; Zhang et al., 2006) identify a new receptor component for Hedgehog, a key morphogen in embryonic development. Many other proteins that bind to Hedgehog in the extracellular matrix or on the cell surface have been identified. In light of these recent discoveries, we discuss how these factors control the stability, transport, reception, and availability of Hedgehog in modulating Hedgehog-mediated responses.     

Low-molecular-weight GTP-binding proteins serving as ADP-ribosylation substrate for ADP-ribosyltransferase from Clostridium botulinum and their relation to phosphoinositides metabolism in thymocytes

ADP-ribosyltransferase from Clostridium botulinum type C strain was found to induce an increase of inositol phosphates (IPs) formation in murine thymocytes membranes. Incubation of electropermeabilized murine thymocytes with the enzyme also caused an increase of IPs formation in the cells. This increase of IPs formation in the enzyme-treated membranes and electropermeabilized cells was dependent on the amount of both NAD and the enzyme, suggesting that the stimulation of phosphoinositide-specific phospholipase C (PLC) was related to ADP-ribosylation of membrane proteins by the enzyme. On the other hand, in calf and murine thymocytes two proteins with the same molecular weight of 21,000 were found to be ADP-ribosylated by the botulinum ADP-ribosyltransferase. A minor ADP-ribosylation substrate was shown by two-dimensional polyacrylamide gel electrophoresis to be G21k, a low-molecular-weight GTP-binding protein (G protein) suggested previously by us to be involved in PLC regulation [Wang, P. et al. (1987) J. Biochem. 102, 1275-1287; (1988) 103, 137-142; and (1989) 105, 461-466], and the other major ADP-ribosylation substrate was identified as a rho A protein. Under the experimental conditions of the IPs formation study, ADP-ribosylation of both G21k and rho A proteins by botulinum ADP-ribosyltransferase in membranes and permeabilized cells was observed. These results suggest that botulinum ADP-ribosyltransferase-induced PLC stimulation in thymocytes is closely correlated with ADP-ribosylation of the low-molecular-weight G proteins.     

Doubling up on microtubule stabilizers: synergistic functions of doublecortin-like kinase and doublecortin in the developing cerebral cortex

Dynamic regulation of neuronal cytoskeletal machinery in response to extracellular cues enables distinct changes in neuronal development in the cerebral cortex. In this issue of Neuron, three related studies on doublecortin-like kinase, a microtubule-associated protein related to doublecortin, by Shu et al., Koizumi et al., and Deuel et al., provide evidence that doublecortin-like kinase is essential for proper neurogenesis, neuronal migration, and axonal wiring.     

Antagonistic activity of Lactobacillus bacteria strains against anaerobic gastrointestinal tract pathogens (Helicobacter pylori, Campylobacter coli, Campylobacter jejuni, Clostridium difficile)]

Antagonistic activity of Lactobacillus strains has been known for some time. This property is connected with production of many active substances by lactobacilli e.g., organic acids and bacteriocin-like substances which interfere with other indigenous microorganisms inhabiting the same ecological niche, including also anaerobic gastrointestinal tract pathogens. Growing interest of clinical medicine in finding new approaches to treatment and prevention of common inflammatory infections of the digestive tract resulted in studies on a possible usage of lactic acid bacteria. Last years, several in vitro and in vivo experiments on antagonism of different Lactobacillus strains against Helicobacter pylori and Clostridium difficile were performed. These observations had been done on already established, well known probiotic Lactobacillus strains. We tested antibacterial activities of Lactobacillus strains isolated from human digestive tract. As indicator bacteria, four species known as anaerobic bacterial etiologic agents of gastroenteric infections: Helicobacter pylori, Campylobacter jejuni, C. coli and Clostridium difficile were used. Some of them were obtained from international collections, others were clinical isolates from specimens taken from patients with different defined gastrointestinal infections. We used a slab method of testing inhibitory activity described in details previously. Following conclusions were drawn from our study: All tested human Lactobacillus strains were able to inhibit the growth of all strains of anaerobic human gastrointestinal pathogens used in this study. Inhibitory activities of tested Lactobacillus strains against Helicobacter pylori, Campylobacter spp., and Clostridium difficile as measured by comparing mean diameters of the inhibition zones were similar. Differences in susceptibility of individual indicator strains of Campylobacter spp. and Clostridium difficile to inhibitory activity of Lactobacillus strains were small. A similar mechanism of inhibition of anaerobic bacteria by lactobacilli is postulated.     

The noodle defense

Macrophages ingest and kill microbes by phagocytosis and delivery to lysosomes. In this issue, Prashar et al. (2013, J. Cell Biol. http://dx.doi.org/10.1083/jcb.201304095) demonstrate that the elongated morphology of filamentous bacteria does not prevent ingestion by macrophages or the fusion of lysosomes, but creates a chimeric, unclosed phagolysosomal compartment whose leakiness blunts the toxicity of lysosomal enzymes, thereby increasing bacterial survival.The tremendous variety of morphologies among microorganisms may allow them to thwart ingestion by the amoeboid cells that patrol pond water and the tissues of animals. Indeed, filament formation by bacteria can limit uptake by phagocytes (Justice et al., 2008). In this issue, Prashar et al. provide mechanistic support for the concept that a filamentous morphology allows bacteria to resist death by phagocytosis.In the normal sequence of events in phagocytosis, outlined in the late nineteenth century and refined by 130 years of microscopy and cell biology, phagocytic cells such as macrophages engage particles by receptor-mediated binding to ligands on microbe surfaces. Receptor signaling leads to the formation of actin-rich, cup-shaped extensions of the cell surface, which enclose particles into membrane-bound intracellular organelles (Swanson, 2008). These phagosomes then mature by progressive fusion with other membranous compartments in the cell, including secretory granules, endosomes, and lysosomes (Flannagan et al., 2009). The mixing of phagosomal and lysosomal contents kills ingested microbes by delivering them into an acidic, hydrolase-rich environment. Ingestion, maturation, and killing have long been considered sequential, nonoverlapping activities.But to be a useful hunting tool or a robust arm of host defense, phagocytosis must overcome microbes in all their various microscopic forms (Fig. 1). To ask how the framework for killing by phagocytosis is altered when a macrophage engages an elongate object, Prashar et al. (2013) examined phagocytosis of filamentous bacteria, which begins after macrophage exploratory movements locate a free filament end (Möller et al., 2012). Once engaged, the macrophage constructed an elongated tubular phagocytic cup comprised of a sleeve of plasma membrane with an actin-rich cuff, which pulled the filament into the cell as if sucking in a long spaghetti noodle. The cup membrane remained contiguous with the plasma membrane until it reached the distal end of the filament, at which point the cup closed into a discrete phagosome inside the cell. Earlier fluorescence microscopy of phagocytosis by macrophages and by Dictyostelium discoideum amebae had shown that membrane phospholipid and protein profiles change even before a phagocytic cup closes into the cell (Botelho et al., 2000; Dormann et al., 2004; Mercanti et al., 2006; Golebiewska et al., 2011). Prashar et al. (2013) identified similar lateral heterogeneity in membrane organization. Remarkably, fusion with early endosomes, late endosomes, and lysosomes began before filaments were fully internalized into the cell, such that the various stages of phagosome maturation were arranged along the length of the tubular phagocytic cups. This extends the earlier studies by showing that the mechanisms which maintain distinct organelle identities do not require organelles to be physically separate from each other inside the cell.Open in a separate windowFigure 1.Extreme phagocytosis. H.S. Jennings’ account (Jennings, 1976) of observations by Rhumbler in 1898: “Ameba verrucosa coiling up and ingesting a filament of Oscillaria. The animal settles upon the middle of an Oscillaria filament, envelopes it, and lengthens out along it (a). Then one end bends over (b), so that a loop is formed in the filament (c). The amoeba then stretches out on the filament again, bends it over anew, and the process is repeated until the filament forms a close coil within the amoeba (c to g).”The delayed closure of elongated phagocytic cups compromised macrophage antimicrobial activities. The actin cuff around the filament formed a tight ring that could limit egress of large dextrans delivered into the phagocytic cup from lysosomes. Nonetheless, while the phagocytic cup membranes remained contiguous with the plasma membrane, protons and lysosomal enzymes leaked out. Complete cup closure was required for phagosome acidification and the full degradative capacity of macrophage defenses. This suggests that adopting a filamentous morphology allows bacteria to lessen the toxicity of microbicidal compounds delivered into the phagosome. Consistent with this idea, Prashar et al. (2013) determined that the survival and growth of Legionella pneumophila in macrophages correlated with filament length.Studies of phagocytosis are undoubtedly important for understanding host defense against infections, but they also provide a vantage point for asking how cells organize cytoplasm for the complexities of microscopic life. Fc receptor–mediated phagocytosis of particles coated with IgG proceeds by a zipper-like mechanism, in which the patterns of IgG ligands on a particle surface guide the distribution of phagocyte signaling that shapes a phagosome (Swanson and Baer, 1995). This localized signaling is modulated by feedback regulation related to the physical properties of the particle. For example, macrophages presented with long, rod-shaped particles coated with IgG respond differently depending on the orientation of their initial contact with those particles. Particles contacted end-on are readily ingested, but particles contacted along their long face are not (Champion and Mitragotri, 2006). This regulation of Fc receptor signaling by surface topology could explain why macrophages ingest filaments only after locating a filament end. But it is a puzzling observation, not least because a macrophage will engage a planar surface coated with IgG as if to engulf it—a response termed “frustrated phagocytosis” (Wright and Silverstein, 1984). Why should phagocytosis proceed on an impossibly large planar surface but not against the side of a rod-shaped particle? Although still unresolved, these fascinating questions about shape-sensing in phagocytosis have been addressed by recent theoretical and experimental studies (Clarke et al., 2010; Dieckmann et al., 2010; Tollis et al., 2010).Another puzzling relationship between Fc receptor signaling and the physical dimensions of the prey concerns the role of phosphatidylinositol 3-kinase (PI3K) in phagocytosis. PI3K is required for phagocytosis of microspheres larger than 3 µm in diameter, but not for phagocytosis of smaller microspheres (Araki et al., 1996; Cox et al., 1999). This suggests that phagocytosis is regulated by a PI3K-dependent feedback related to particle size. How does that work? It could be that PI3K relieves a feedback inhibition of Fc receptor signaling that begins only after some delay, such that it becomes rate-limiting only for the phagocytosis of larger particles that take more time to ingest. Prashar et al. (2013) excluded that possibility by examining the effects of PI3K inhibition on the phagocytosis of filaments. In the presence of the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, phagocytosis of IgG-opsonized sheep erythrocytes was inhibited but the phagocytosis of filaments was not. This suggests that formation of the narrow aperture for sucking in a noodle occurs below the PI3K-dependent size threshold. Moreover, once a phagocytic response was initiated, it continued for as long as necessary to ingest the filament, showing that PI3K-dependent regulation of phagocytosis is spatial rather than temporal. The 3′ phosphoinositide products of PI3K may be part of a system for gauging the three-dimensional distribution of phagocytic receptor signaling, a mechanism of spatial integration that regulates cellular commitment to phagocytosis (Zhang et al., 2010). In the battles with the exotic geometries of microbial life, it may be necessary for a phagocyte to decide whether a particle is small enough to eat or should instead be held outside and engaged as extracellular prey. Accordingly, the narrow end of a filament presents a stimulus that is below the size threshold for PI3K-dependent regulation, so the macrophage begins slurping it in, with a considerable amount of lysosomal spittle dribbling out.     

Burkholderia cenocepacia disrupts host cell actin cytoskeleton by inactivating Rac and Cdc42

Burkholderia cenocepacia, a member of the Burkholderia cepacia complex, is an opportunistic pathogen that causes devastating infections in patients with cystic fibrosis. The ability of B. cenocepacia to survive within host cells could contribute significantly to its virulence in immunocompromised patients. In this study, we explored the mechanisms that enable B. cenocepacia to survive inside macrophages. We found that B. cenocepacia disrupts the actin cytoskeleton of infected macrophages, drastically altering their morphology. Submembranous actin undergoes depolymerization, leading to cell retraction. The bacteria perturb actin architecture by inactivating Rho family GTPases, particularly Rac1 and Cdc42. GTPase inactivation follows internalization of viable B. cenocepacia and compromises phagocyte function: macropinocytosis and phagocytosis are markedly inhibited, likely impairing the microbicidal and antigen‐presenting capability of infected macrophages. The type VI secretion system is essential for the bacteria to elicit these changes. This is the first report demonstrating inactivation of Rho family GTPases by a member of the B. cepacia complex.     

Disruption of bacterial quorum sensing by other organisms

Higher plants and algae produce compounds that mimic quorum sensing: signals used by bacteria to regulate the expression of many genes and behaviors. Similarly, various bacteria can stimulate, inhibit or inactivate quorum sensing in other bacteria. These discoveries offer new opportunities to manipulate bacterial quorum sensing in applications relevant to medicine, agriculture and the environment.     

Genome-Wide Analysis of Gene Expression in Stationary Phase and Genetic Characterization of Stationary-Phase-Dependent Halocin Gene Expression in the Haloarchaeon Haloferax mediterranei

The stationary phase of microbial growth is a very complex state regulated by various environmental and physiological factors.An intensive study of stationary phase could promote a comprehensive understanding of the complete life cycle of microorganisms,and may provide important insights into their adaptation to harsh and nutrient-depleted conditions.Although the underlying mechanisms have been well-studied in bacteria and yeasts (Herman,2002;Navarro Llorens et al.,2010),less is known about this growth phase in archaea yet.The haloarchaeon Haloferax mediterranei has served as a good model for studying haloarchaeal physiology and metabolism for several decades because of its accelerated growth,remarkable metabolic ability and genomic stability (Han et al.,2012).During stationary phase,H.mediterranei can produce halocin H4 (Cheung et al.,1997),synthesize gas vesicles (J(a)ger et al.,2002),secrete extracellular polysaccharide (Antón et al.,1988) and accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)(Cai et al.,2012).Due to these specific features,we selected H.mediterranei as a model system to investigate the archaeal gene expression and regulation during the stationary phase.     

ADP-ribosylation of small GTP-binding proteins by Bacillus cereus.

A human pathogenic strain of Bacillus cereus produces an exoenzyme which selectively ADP-ribosylates 20-25 kDa GTP-binding proteins in platelet membranes. Pre-ADP-ribosylation of rho proteins of human platelet membranes with Clostridium botulinum exoenzyme C3 or Clostridium limosum exoenzyme inhibits subsequent ADP-ribosylation by the exoenzyme from B. cereus indicating similar substrate specificity of the transferases. The ADP-ribosyltransferase from B. cereus reveals no immunological cross-reactivity with C. botulinum C3 and C. limosum exoenzyme.     

Brucella "hitches a ride" with autophagy

Autophagy involves lysosomal-mediated degradation of cellular components and contributes to host immunity. Some pathogens avoid autophagy-mediated killing, while others exploit it to acquire host cell nutrients. Starr et?al. reveal that the intracellular bacterial pathogen Brucella abortus can "hitch a ride" with autophagy, subverting autophagy machinery to spread from cell to cell (Starr et?al., 2012).     

Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.     

Role of several physico-chemical surface properties of bacteria in the machanism of the initial phases of phagocytosis]

An attempt was made on E. coli pattern to reveal the role played by the surface charge and the extent of the surface hydration in the mechanism of the initial phagocytosis phases (attraction and submersion). Phagocytosis experiments with washed bacteria and washed rabbit leukocytes demonstrated a marked direct dependence of the electrophoretic velocities of escherichia and the intensity of their phagocytic ingestion, and a strong reverse relationship between the extent of hydration and the phagocytic activity. Treatment of bacteria with rabbit plasma with subsequent washing led to a significant reduction of hydration and to an insignificant reduction of the electrophoretic velocities with parallel increase of the phagocyte activity. Correlation between the phagocytosis intensity and the acquired physico-chemical properties of bacteria became weak.     

Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.

The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)