Soft-x-ray resonant scattering from V/Fe (001) magnetic superlattices

The magnetic properties of Fe and V in (n ML V / 5 ML Fe) (001) superlattices (n = 1, 2, and 5 ML) have been investigated by measuring the magnetization dependent scattering of elliptically polarized soft x rays at various scattering angles and for photon energies across the 2p x-ray-absorption edges of both Fe and V. A quantitative estimate of the magnetic moment induced on vanadium is obtained from the analysis of the resonant scattering data. [S0163-1829(99)50842-4]

Flagellin-Deficient Legionella Mutants Evade Caspase-1- and Naip5-Mediated Macrophage Immunity

Macrophages from C57BL/6J (B6) mice restrict growth of the intracellular bacterial pathogen Legionella pneumophila. Restriction of bacterial growth requires caspase-1 and the leucine-rich repeat-containing protein Naip5 (Birc1e). We identified mutants of L. pneumophila that evade macrophage innate immunity. All mutants were deficient in expression of flagellin, the primary flagellar subunit, and failed to induce caspase-1-mediated macrophage death. Interestingly, a previously isolated flagellar mutant (fliI) that expresses, but does not assemble, flagellin did not replicate in macrophages, and induced macrophage death. Thus, flagellin itself, not flagella or motility, is required to initiate macrophage innate immunity. Immunity to Legionella did not require MyD88, an essential adaptor for toll-like receptor 5 (TLR5) signaling. Moreover, flagellin of Legionella and Salmonella induced cytotoxicity when delivered to the macrophage cytosol using Escherichia coli as a heterologous host. It thus appears that macrophages sense cytosolic flagellin via a TLR5-independent pathway that leads to rapid caspase-1-dependent cell death and provides defense against intracellular bacterial pathogens

Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter

Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α5β1 integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis

Structural insight into the membrane targeting domain of the Legionella deAMPylase SidD

AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (??loop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidD??loop phenocopied a L. pneumophila ??sidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection

Inhibition of Host Vacuolar H+-ATPase Activity by a Legionella pneumophila Effector

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae

Innate Immune Recognition of Yersinia pseudotuberculosis Type III Secretion

Specialized protein translocation systems are used by many bacterial pathogens to deliver effector proteins into host cells that interfere with normal cellular functions. How the host immune system recognizes and responds to this intrusive event is not understood. To address these questions, we determined the mammalian cellular response to the virulence-associated type III secretion system (T3SS) of the human pathogen Yersinia pseudotuberculosis. We found that macrophages devoid of Toll-like receptor (TLR) signaling regulate expression of 266 genes following recognition of the Y. pseudotuberculosis T3SS. This analysis revealed two temporally distinct responses that could be separated into activation of NFκB- and type I IFN-regulated genes. Extracellular bacteria were capable of triggering these signaling events, as inhibition of bacterial uptake had no effect on the ensuing innate immune response. The cytosolic peptidoglycan sensors Nod1 and Nod2 and the inflammasome component caspase-1 were not involved in NFκB activation following recognition of the Y. pseudotuberculosis T3SS. However, caspase-1 was required for secretion of the inflammatory cytokine IL-1β in response to T3SS-positive Y. pseudotuberculosis. In order to characterize the bacterial requirements for induction of this novel TLR-, Nod1/2-, and caspase-1-independent response, we used Y. pseudotuberculosis strains lacking specific components of the T3SS. Formation of a functional T3SS pore was required, as bacteria expressing a secretion needle, but lacking the pore-forming proteins YopB or YopD, did not trigger these signaling events. However, nonspecific membrane disruption could not recapitulate the NFκB signaling triggered by Y. pseudotuberculosis expressing a functional T3SS pore. Although host cell recognition of the T3SS did not require known translocated substrates, the ensuing response could be modulated by effectors such as YopJ and YopT, as YopT amplified the response, while YopJ dampened it. Collectively, these data suggest that combined recognition of the T3SS pore and YopBD-mediated delivery of immune activating ligands into the host cytosol informs the host cell of pathogenic challenge. This leads to a unique, multifactorial response distinct from the canonical immune response to a bacterium lacking a T3SS

Extensive recombination events and horizontal gene transfer shaped the Legionella pneumophila genomes

<p>Abstract</p> <p>Background</p> <p><it>Legionella pneumophila </it>is an intracellular pathogen of environmental protozoa. When humans inhale contaminated aerosols this bacterium may cause a severe pneumonia called Legionnaires' disease. Despite the abundance of dozens of <it>Legionella </it>species in aquatic reservoirs, the vast majority of human disease is caused by a single serogroup (Sg) of a single species, namely <it>L. pneumophila </it>Sg1. To get further insights into genome dynamics and evolution of Sg1 strains, we sequenced strains Lorraine and HL 0604 1035 (Sg1) and compared them to the available sequences of Sg1 strains Paris, Lens, Corby and Philadelphia, resulting in a comprehensive multigenome analysis.</p> <p>Results</p> <p>We show that <it>L. pneumophila </it>Sg1 has a highly conserved and syntenic core genome that comprises the many eukaryotic like proteins and a conserved repertoire of over 200 Dot/Icm type IV secreted substrates. However, recombination events and horizontal gene transfer are frequent. In particular the analyses of the distribution of nucleotide polymorphisms suggests that large chromosomal fragments of over 200 kbs are exchanged between <it>L. pneumophila </it>strains and contribute to the genome dynamics in the natural population. The many secretion systems present might be implicated in exchange of these fragments by conjugal transfer. Plasmids also play a role in genome diversification and are exchanged among strains and circulate between different <it>Legionella </it>species.</p> <p>Conclusion</p> <p>Horizontal gene transfer among bacteria and from eukaryotes to <it>L. pneumophila </it>as well as recombination between strains allows different clones to evolve into predominant disease clones and others to replace them subsequently within relatively short periods of time.</p

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described