Host Cell-catalyzed S-Palmitoylation Mediates Golgi Targeting of the Legionella Ubiquitin Ligase GobX.

The facultative intracellular pathogen Legionella pneumophila, the causative agent of Legionnaires disease, infects and replicates within human alveolar macrophages. L. pneumophila delivers almost 300 effector proteins into the besieged host cell that alter signaling cascades and create conditions that favor intracellular bacterial survival. In order for the effectors to accomplish their intracellular mission, their activity needs to be specifically directed toward the correct host cell protein or target organelle. Here, we show that the L. pneumophila effector GobX possesses E3 ubiquitin ligase activity that is mediated by a central region homologous to mammalian U-box domains. Furthermore, we demonstrate that GobX exploits host cell S-palmitoylation to specifically localize to Golgi membranes. The hydrophobic palmitate moiety is covalently attached to a cysteine residue at position 175, which is part of an amphipathic α-helix within the C-terminal region of GobX. Site-directed mutagenesis of cysteine 175 or residues on the hydrophobic face of the amphipathic helix strongly attenuated palmitoylation and Golgi localization of GobX. Together, our study provides evidence that the L. pneumophila effector GobX exploits two post-translational modification pathways of host cells, ubiquitination and S-palmitoylation.

InlP, a New Virulence Factor with Strong Placental Tropism.

Intrauterine infection is a major detriment for maternal-child health and occurs despite local mechanisms that protect the maternal-fetal interface from a wide variety of pathogens. The bacterial pathogen Listeria monocytogenes causes spontaneous abortion, stillbirth, and preterm labor in humans and serves as a model for placental pathogenesis. Given the unique immunological environment of the maternal-fetal interface, we hypothesized that virulence determinants with placental tropism are required for infection of this tissue. We performed a genomic screen in pregnant guinea pigs that led to the identification of 201 listerial genes important for infection of the placenta but not maternal liver. Among these genes was lmrg1778 (lmo2470), here named inlP, predicted to encode a secreted protein that belongs to the internalin family. InlP is conserved in virulent L. monocytogenes strains but absent in Listeria species that are nonpathogenic for humans. The intracellular life cycle of L. monocytogenes deficient in inlP (ΔinlP) was not impaired. In guinea pigs and mice, InlP increased the placental bacterial burden by a factor of 3 log10 while having only a minor role in other maternal organs. Furthermore, the ΔinlP strain was attenuated in intracellular growth in primary human placental organ cultures and trophoblasts. InlP is a novel virulence factor for listeriosis with a strong tropism for the placenta. This virulence factor represents a tool for the development of new modalities to prevent and treat infection-related pregnancy complications.

Periplasmic superoxide dismutase SodCI of Salmonella binds peptidoglycan to remain tethered within the periplasm.

Salmonellae survive and propagate in macrophages to cause serious systemic disease. Periplasmic superoxide dismutase plays a critical role in this survival by combating phagocytic superoxide. Salmonella Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Although both proteins are produced during infection, only SodCI is functional in the macrophage phagosome. We have previously shown that SodCI, relative to SodCII, is both protease resistant and tethered within the periplasm and that either of these properties is sufficient to allow a SodC to protect against phagocytic superoxide. Tethering is defined as remaining cell-associated after osmotic shock or treatment with cationic antimicrobial peptides. Here we show that SodCI non-covalently binds peptidoglycan. SodCI binds to Salmonella and Bacillus peptidoglycan, but not peptidoglycan from Staphylococcus. Moreover, binding can be inhibited by a diaminopimelic acid containing tripeptide, but not a lysine containing tripeptide, showing that the protein recognizes the peptide portion of the peptidoglycan. Replacing nine amino acids in SodCII with the corresponding residues from SodCI confers tethering, partially delineating an apparently novel peptidoglycan binding domain. These changes in sequence increase the affinity of SodCII for peptidoglycan fragments to match that of SodCI and allow the now tethered SodCII to function during infection.

Effect of Si/Al molar ratio and curing temperatures on the immobilization of radioactive borate waste in metakaolin-based geopolymer waste form.

Immobilization of radioactive borate waste (RBW) using a geopolymer with a high Si/Al ratio has been challenging because boron-silicon networks lower the compressive strength and delay the setting time. In this study, metakaolin-based geopolymer waste form to immobilize simulant RBW was fabricated using different Si/Al ratios (1.0-1.4) and curing temperatures (26 and 60 â„ƒ). The 7-day compressive strength results revealed that a certain amount of silicon and an elevated curing temperature are required to achieve high compressive strength and waste loading. Following waste acceptance criteria tests, all geopolymers exhibited compressive strengths higher than 3.445 MPa. The leachability index of boron was higher than 6.0, and the leaching mechanism was identified as diffusion. No significant structural changes in the geopolymer were observed after thermal cycling and gamma irradiation tests. The physically bound or unincorporated RBW was leached out of the geopolymer during water immersion and leaching tests; however, boron, which was chemically connected with silicon, was present as an inert phase together with a geopolymer binder. Consequently, immobilizing RBW using a geopolymer with a low Si/Al ratio (1.4) is beneficial in terms of RBW loading and structural durability.

Development of geopolymer waste form for immobilization of radioactive borate waste.

Radioactive borate waste containing a high concentration of boron (B) is problematic to be solidified using cement because soluble borate such as boric acid hinders the hydration reaction. In this study, borate waste was used as a raw material for metakaolin-based geopolymer according to the characteristic that B replaces a part of Si. Geopolymers using KOH alkaline activator (K-geopolymers) showed higher compressive strength than geopolymers using NaOH alkaline activator (Na-geopolymer). In addition, the compressive strength increased proportionally to the Si/(Al+B) ratio regardless of the alkaline cation species. These variations in compressive strength might be due to the viscosity of the geopolymer mixture, atomic size of alkaline cations, and the increase in Si content. The characteristic analyses (XRD, FT-IR, and solid state 11B MAS NMR) indicated that B was incorporated into the geopolymer structure. Thus, the K-geopolymer has a dense and homogeneous microstructure. In a semi-dynamic leaching test, less B leached from the geopolymers compared to the cement waste form. Consequently, borate waste can be solidified using metakaolin-based geopolymer, and the use of a KOH alkaline activator is advantageous in terms of mechanical property and structural durability.

Setting Behavior and Phase Evolution on Heat Treatment of Metakaolin-Based Geopolymers Containing Calcium Hydroxide.

The setting behavior of geopolymers is affected by the type of source materials, alkali activators, mix formulations, and curing conditions. Calcium hydroxide is known to be an effective additive to shorten the setting period of geopolymers. However, there is still room for improvement in the understanding of the effect of calcium hydroxide on the setting and phase evolution of geopolymers. In this study, the setting behavior and phase evolution of geopolymer containing calcium hydroxide were investigated by XRD analysis. The setting time of the geopolymer was inconsistently shortened as the amount of calcium hydroxide increased. A low calcium hydroxide dose of up to 2% of the total mix weight could contribute to the enhancement of compressive strength of geopolymers besides a fast-setting effect. The C-S-H gel is rapidly precipitated at the early stage of reaction in geopolymers containing high calcium hydroxide with some of the calcium hydroxide remaining intact. The ex-situ high-temperature XRD analysis and Rietveld refinement results revealed that geopolymer and C-S-H gel transformed into Si-rich nepheline and wollastonite, respectively. The wollastonite was also observed in heat-treated geopolymers with a low calcium hydroxide dose. It is believed that C-S-H gel can be precipitated along with geopolymers regardless of how much calcium hydroxide is added.

A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila.

Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

Protecting against antimicrobial effectors in the phagosome allows SodCII to contribute to virulence in Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium replicates in macrophages, where it is subjected to antimicrobial substances, including superoxide, antimicrobial peptides, and proteases. The bacterium produces two periplasmic superoxide dismutases, SodCI and SodCII. Although both are expressed during infection, only SodCI contributes to virulence in the mouse by combating phagocytic superoxide. The differential contribution to virulence is at least partially due to inherent differences in the SodCI and SodCII proteins that are independent of enzymatic activity. SodCII is protease sensitive, and like other periplasmic proteins, it is released by osmotic shock. In contrast, SodCI is protease resistant and is retained within the periplasm after osmotic shock, a phenomenon that we term "tethering." We hypothesize that in the macrophage, antimicrobial peptides transiently disrupt the outer membrane. SodCII is released and/or phagocytic proteases gain access to the periplasm, and SodCII is degraded. SodCI is tethered within the periplasm and is protease resistant, thereby remaining to combat superoxide. Here we test aspects of this model. SodCII was released by the antimicrobial peptide polymyxin B or a mouse macrophage antimicrobial peptide (CRAMP), while SodCI remained tethered within the periplasm. A Salmonella pmrA constitutive mutant no longer released SodCII in vitro. Moreover, in the constitutive pmrA background, SodCII could contribute to survival of Salmonella during infection. SodCII also provided a virulence benefit in mice genetically defective in production of CRAMP. Thus, consistent with our model, protecting the outer membrane against antimicrobial peptides allows SodCII to contribute to virulence in vivo. These data also suggest direct in vivo cooperative interactions between macrophage antimicrobial effectors.

Beneficial Use of MIBC in Metakaolin-Based Geopolymers to Improve Flowability and Compressive Strength.

Superplasticizers (cement concrete water reducers) are applied to improve the flowability of calcium-rich, alkali-activated materials, with inconsistent results. However, superplasticizer applications are limited in metakaolin-based geopolymers. The possibility of using polycarboxylate superplasticizers and methyl isobutyl carbinol (MIBC) to ameliorate the flowability of metakaolin-based geopolymers was investigated. The ratio of metakaolin, fumed silica, NaOH or KOH, and water in geopolymers at a Na2O or K2O:Al2O3:SiO2:H2O ratio = 1:1:4:10 or 1:1:4:11 was maintained in the formulations. In this study, ether- or ester-based polycarboxylate superplasticizers did not improve the workability of fresh metakaolin-based Na-geopolymers. A low MIBC dose (0.5 wt.% of metakaolin) improved the flowability by 19% and additionally increased the 7-day compressive strength by 22% from 68 to 83 MPa for plain Na-geopolymers. The entrained fine froths produced by adding MIBC during mixing likely reduced friction between metakaolin particles, and the slurry became more workable. Hence, the geopolymer mixture with an improved flowability became more homogenous, which ensured more extensive metakaolin dissolution and hydrolysis. A low MIBC dose could be effective for Na-geopolymers with dual benefits of improved workability and enhanced compressive strength.

Advanced characterization of IGCC slag by automated SEM-EDS analysis.

Integrated gasification combined cycle (IGCC) is a highly efficient method for producing electricity but discharges a byproduct in the form of a glassy slag, similar to other electricity generation operations. Several technologies for recycling IGCC slag have been developed thus far, although the results obtained are not promising or universally applicable. We quantitatively characterized an IGCC slag by using various testing methods, including an automated scanning electron microscopy-energy dispersive spectrometry (SEM-EDS) system, to recognize its potential for recycling. The IGCC slag did not contain free CaO, and the absence of free lime would address a concern of volumetric expansion during hydration. Automated SEM-EDS analysis revealed that approximately 98% of the IGCC slag particles consisted of calcium-rich aluminosilicate materials. Obvious differences in the concentrations of Si, Al, and Ca between the amorphous phases and the average chemical bulk were recognized. The chemical composition of the amorphous Si-Al-Ca phases was similar to that of Class C fly ash, while the average bulk composition of the IGCC slag was in between that of Class C and Class F fly ashes. Considering this discrepancy, understanding the dissolution mechanism of the reactive amorphous fraction as well as an exact assessment of the reaction products based on the role of Ca in alkali-activated materials provides a new approach for the valorization of IGCC slag.

DsbL and DsbI contribute to periplasmic disulfide bond formation in Salmonella enterica serovar Typhimurium.

Disulfide bond formation in periplasmic proteins is catalysed by the DsbA/DsbB system in most Gram-negative bacteria. Salmonella enterica serovar Typhimurium also encodes a paralogous pair of proteins to DsbA and DsbB, DsbL and DsbI, respectively, downstream of a periplasmic arylsulfate sulfotransferase (ASST). We show that DsbL and DsbI function as a redox pair contributing to periplasmic disulfide bond formation and, as such, affect transcription of the Salmonella pathogenicity island 1 (SPI1) type three secretion system genes and activation of the RcsCDB system, as well as ASST activity. In contrast to DsbA/DsbB, however, the DsbL/DsbI system cannot catalyse the disulfide bond formation required for flagellar assembly. Phylogenic analysis suggests that the assT dsbL dsbI genes are ancestral in the Enterobacteriaceae, but have been lost in many lineages. Deletion of assT confers no virulence defect during acute Salmonella infection of mice.

Unexpected Errors in Gas Chromatographic Analysis of Methane Production by Thermophilic Bacteria.

Unexpected errors in methane measurement by gas chromatography occurred when samples at thermophilic temperatures were analyzed. With a standard curve prepared at room temperature (25 degrees C), stoppered bottles incubated and sampled at 37 to 85 degrees C showed more methane upon analysis than bottles incubated at 25 degrees C: values at 50, 63, and 85 degrees C were 109, 126, and 125%, respectively, of the 25 degrees C value. All variation between 4 and 50 degrees C can be explained by the temperature difference between culture bottle and sampling syringe, and the variation of methane concentration can be predicted by the gas law. Between 50 and 63 degrees C, there was a more dramatic rise than predicted by theory. These variations are important to consider if thermophilic methane production is to be measured accurately. Methods to avoid errors are discussed.

Enhanced immune responses of foot-and-mouth disease vaccine using new oil/gel adjuvant mixtures in pigs and goats.

The immunity and protective capability produced by vaccines can vary remarkably according to the kinds of adjuvants being used. In the case of foot-and-mouth disease (FMD) vaccines in pigs, only oil-adjuvant vaccines have been used, and these tend to show lower immunity in pigs than in cattle. New adjuvants for these vaccines are therefore needed. We made different experimental FMD vaccines using new adjuvants (ISA 201, Carbigen, Emulsigen-D) and well-known adjuvants (ISA 206, aluminum hydroxide gel) and then conducted tests to compare the enhancement in pig immunity. More effective immune responses and protection against challenge were observed with the new adjuvants Emulsigen-D and ISA 201 compared to existing adjuvants. In the case of dairy goats, a mixture of Emulsigen-D and aluminum hydroxide gel produced rapid neutralizing antibody responses that were similar to results from tests conducted with pigs.

Structural properties of periplasmic SodCI that correlate with virulence in Salmonella enterica serovar Typhimurium.

Salmonella enterica strains survive and propagate in macrophages by both circumventing and resisting the antibacterial effectors normally delivered to the phagosome. An important aspect of Salmonella resistance is the production of periplasmic superoxide dismutase to combat phagocytic superoxide. S. enterica serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases: SodCI and SodCII. Both enzymes are produced during infection, but only SodCI contributes to virulence in the animal. Although 60% identical to SodCII at the amino acid level with very similar enzymatic properties, SodCI is dimeric, protease resistant, and tethered within the periplasm via a noncovalent interaction. In contrast, SodCII is monomeric and protease sensitive and is released from the periplasm normally by osmotic shock. We have constructed an enzymatically active monomeric SodCI enzyme by site-directed mutagenesis. The resulting protein was released by osmotic shock and sensitive to protease and could not complement the loss of wild-type dimeric SodCI during infection. To distinguish which property is most critical during infection, we cloned and characterized related SodC proteins from a variety of bacteria. Brucella abortus SodC was monomeric and released by osmotic shock but was protease resistant and could complement SodCI in the animal. These data suggest that protease resistance is a critical property that allows SodCI to function in the harsh environment of the phagosome to combat phagocytic superoxide. We propose a model to account for the various properties of SodCI and how they contribute to bacterial survival in the phagosome.