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Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface. Such complexes are observed when GBP1 binds lipopolysaccharide (LPS) from Shigella and Salmonella and further recruits GBP2-4. Here, we compared GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and S. flexneri. Francisella novicida was coated by GBP1 and GBP2 and to a lower extent by GBP4 in human macrophages. Contrary to S. flexneri, F. novicida was not targeted by GBP3, a feature independent of T6SS effectors. Multiple GBP1 features were required to promote targeting to F. novicida while GBP1 targeting to S. flexneri was much more permissive to GBP1 mutagenesis suggesting that GBP1 has multiple domains that cooperate to recognize F. novicida atypical LPS. Altogether our results indicate that the repertoire of GBPs recruited onto specific bacteria is dictated by GBP-specific features and by specific bacterial factors that remain to be identified.

Group I chaperonins are a highly conserved family of essential proteins that self-assemble into molecular nanoboxes that mediate the folding of cytoplasmic proteins in bacteria and organelles. GroEL, the chaperonin of Escherichia coli, is the archetype of the family. Protein folding-independent functions have been described for numerous chaperonins, including HtpB, the chaperonin of the bacterial pathogen Legionella pneumophila. Several protein folding-independent functions attributed to HtpB are not shared by GroEL, suggesting that differences in the amino acid (aa) sequence between these two proteins could correlate with functional differences. GroEL and HtpB differ in 137 scattered aa positions. Using the Evolutionary Trace (ET) bioinformatics method, site-directed mutagenesis, and a functional reporter test based upon a yeast-two-hybrid interaction with the eukaryotic protein ECM29, it was determined that out of those 137 aa, ten (M68, M212, S236, K298, N507 and the cluster AEHKD in positions 471-475) were involved in the interaction of HtpB with ECM29. GroEL was completely unable to interact with ECM29, but when GroEL was modified at those 10 aa positions, to display the HtpB aa, it acquired a weak ability to interact with ECM29. This constitutes proof of concept that the unique functional abilities of HtpB can be mapped to specific aa positions.

Background Pseudomonas aeruginosa forms antibiotic-resistant biofilms that are responsible for the treatment failure or relapses of the bacterial infections in the lungs of patients with cystic fibrosis (CF). The alginate lyases that target extracellular polysaccharide alginate of P. aeruginosa biofilms are promising therapeutic candidates for treatment of P. aeruginosa biofilm infections. Methods Immunofluorescent staining and thin layer chromatography were used to demonstrate the alginolytic activity of the alginate lyase enzyme (AlyP1400) purified from a marine Pseudoalteromonas bacterium. Anti-biofilm activities of AlyP1400 were tested alone or in combination with antibiotics on the biofilms of a mucoid Pseudomonas aeruginosa clinical isolate CF27 that were cultivated in 96-well plates and a flow cell. Results We showed that AlyP1400 facilitated antibiotic activities to eliminate CF27 biofilms. The combination of AlyP1400 with antibiotics reduced the biofilm biomass and boosted bactericidal activity of antibiotics. Importantly, we demonstrated that the enzymatic activity of AlyP1400 was required for its biofilm disruption activity and its synergy with antibiotics to eradicate biofilm cells. Conclusion This work shed new light on the potential mechanisms of the therapeutic activity for the combinational use of alginate lyase and antibiotics to treat P. aeruginosa infections in CF lungs or other P. aeruginosa biofilm-related infections.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ stimulated cells guanylate binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4. Caspase-4 activation is hierarchically controlled by GBPs; GBP1 initiates platform assembly, GBP2 and GBP4 control caspase-4 recruitment, whereas GBP3 governs caspase-4 activation. In response to cytosol-invading bacteria, activation of caspase-4 through the GBP platform is essential to induce gasdermin-D dependent pyroptosis and processing of interleukin-18, thereby destroying the replicative niche for intracellular bacteria and alerting neighboring cells, respectively. Caspase-11 and GBPs epistatically protect mice against lethal bacterial challenge. Multiple antagonists of the pathway encoded by Shigella flexneri, a cytosol-adapted bacterium, provide compelling evolutionary evidence for the importance of the GBP-Caspase-4 pathway in anti-bacterial defense.

Cdk8 of the RNA polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55 and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation., Horvath, Hawe et al. examine regulators of Cdk8-dependent Gal4 activity in a screen for defects in the LTA response of gal3 yeast. They identify TORC1 components tor1 and tco89 and the downstream PP2A/Cdc55 phosphatase as regulators of Gal4. Additionally, genetic analysis indicates that mutations in hom3, which encodes aspartate kinase, produce defects in TOR signaling. These results demonstrate that regulation of Gal4 by phosphorylation is modulated by the TOR nutritional signaling pathway through the activity of PP2A/Cdc55.

Defective autophagy is associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn’s disease by enhancing innate immunity in macrophages. However, autophagy also mediates clearance of intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in antimicrobial immunity. In this study, we found that loss of Atg16l1 in macrophages enhanced the killing of virulent Shigella flexneri (S.flexneri), an enteric bacterium that resides within the cytosol by escaping all membrane-bound compartments. Quantitative multiplexed proteomics revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which also promoted S.flexneri killing. Consistently, myeloid cell-specific deletion of Atg16l1 accelerated bacterial clearance in vivo. Finally, pharmacological modulation of oxidative stress by suppression of cysteine import conferred enhanced microbicidal properties to wild type macrophages. These findings demonstrate that control of oxidative stress by ATG16L1 regulates antimicrobial immunity against intracellular pathogens.Impact statementMaculins et al utilize multiplexed mass spectrometry to show that loss of the autophagy gene Atg16l1 in macrophages enhances antimicrobial immunity against intracellular pathogens via the oxidative stress response.

Cdk8 of the RNA Polymerase II mediator complex regulates genes by phosphorylating sequence specific transcription factors. Despite conserved importance for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full induction of the yeastGALgenes requires phosphorylation of Gal4 by Cdk8, and we exploited this requirement for growth ofgal3yeast on galactose to identify mutants affecting Cdk8 activity. Several mutants from the screen produced defects in TOR signaling. A mutant designatedgalfour throttle (gft) 1,gft1, was identified as an allele ofhom3, encoding aspartokinase. Defects ingft1/hom3caused hypersensitivity to rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations oftor1ortco89, encoding TORC1 components, also preventedGALexpression ingal3yeast, andtco89was determined to be allelic togft7. Disruption ofcdc55, encoding a subunit of PP2A regulated by TOR signaling, suppressed the effect ofgft1/hom3, gft7/tco89, andtor1mutations onGALexpression ingal3yeast, but not ofcdk8/srb10disruptions or Gal4 S699A mutation. Mutations ofgft1/hom3andtor1did not affect kinase activity of Cdk8in vitro, but caused loss of Gal4 phosphorylationin vivo. These observations demonstrate that TOR signaling regulatesGALinduction through the activity of PP2A/ Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

Recall of microbial-contaminated food products is an important intervention in preventing the transmission of foodborne illness. Here, we summarize the number and nature of foods recalled as a result of microbial contamination, classified by the Canadian Food Inspection Agency, for the period 1 January 2000 through 31 December 2017. A total of 10,432 food products were recalled from 2,094 recall events in Canada because of microbial contamination during this period. The meat, meat products and poultry category, followed by fishery and seafood products and nuts and edible seeds, contained the food products most commonly associated with microbial contamination. Most microbial-contaminated food products reported were recalled because of the presence bacterial pathogens. Salmonella contamination was responsible for the largest number of recall events, whereas Listeria monocytogenes contamination accounted for the greatest number of food products recalled because of microbial contamination. L. monocytogenes contamination was also most commonly associated with major food recall events, although records may be inflated because of an invested effort to prevent future L. monocytogenes outbreaks following a 2008 deli meat recall. The findings and data we present in this study will support future surveillance and analysis of microbial-contaminated food recalls in Canada.

Intracellular trafficking pathways in eukaryotic cells are essential to maintain organelle identity and structure, and to regulate cell communication with its environment. Shigella flexneri invades and subverts the human colonic epithelium by the injection of virulence factors through a type 3 secretion system (T3SS). In this work, we report the multiple effects of two S. flexneri effectors, IpaJ and VirA, which target small GTPases of the Arf and Rab families, consequently inhibiting several intracellular trafficking pathways. IpaJ and VirA induce large-scale impairment of host protein secretion and block the recycling of surface receptors. Moreover, these two effectors decrease clathrin-dependent and -independent endocytosis. Therefore, S. flexneri infection induces a global blockage of host cell intracellular transport, affecting the exchange between cells and their external environment. The combined action of these effectors disorganizes the epithelial cell polarity, disturbs epithelial barrier integrity, promotes multiple invasion events, and enhances the pathogen capacity to penetrate into the colonic tissue in vivo.

Pseudomonas aeruginosa biofilms are typically associated with the chronic lung infection of cystic fibrosis (CF) patients and represent a major challenge for treatment. This opportunistic bacterial pathogen secretes alginate, a polysaccharide that is one of the main components of its biofilm. Targeting this major biofilm component has emerged as a tempting therapeutic strategy for tackling biofilm-associated bacterial infections. The enormous potential in genetic diversity of the marine microbial community make it a valuable resource for mining activities responsible for a broad range of metabolic processes, including the alginolytic activity responsible for degrading alginate. A collection of 36 bacterial isolates were purified from marine water based on their alginolytic activity. These isolates were identified based on their 16S rRNA gene sequences. Pseudoalteromonas sp. 1400 showed the highest alginolytic activity and was further confirmed to produce the enzyme alginate lyase. The purified alginate lyase (AlyP1400) produced by Pseudoalteromonas sp. 1400 showed a band of 23 KDa on a protein electrophoresis gel and exhibited a bifunctional lyase activity for both poly-mannuronic acid and poly-glucuronic acid degradation. A tryptic digestion of this gel band analyzed by liquid chromatography-tandem mass spectrometry confirmed high similarity to the alginate lyases in polysaccharide lyase family 18. The purified alginate lyase showed a maximum relative activity at 30 &deg, C at a slightly acidic condition. It decreased the sodium alginate viscosity by over 90% and reduced the P. aeruginosa (strain PA14) biofilms by 69% after 24 h of incubation. The combined activity of AlyP1400 with carbenicillin or ciprofloxacin reduced the P. aeruginosa biofilm thickness, biovolume and surface area in a flow cell system. The present data revealed that AlyP1400 combined with conventional antibiotics helped to disrupt the biofilms produced by P. aeruginosa and can be used as a promising combinational therapeutic strategy.

The purpose of this work was to develop a method of determining farfield principal stress magnitude and orientation using biaxial stress measurements made in a series of bore holes. Two novel developments were employed: one was a method for relating biaxial measurements from three bore holes to the triaxial principal stress field; the other was a mea­ surement technique that fully defines the biaxial stress field around a bore hole, independent of rock properties. To determine the triaxial principal stress field, a series of simultaneous equations describing biaxial measurements in terms of a random baseline coordinate system was developed. A least squares regression was then utilized to determine the coordinate system's stress field. A Newton-Raphson reduction was subse­ quently performed to define the orientation and magnitude of the prin­ cipal stress field. The biaxial stress measurement technique employs two loading mechanisms and ultrasonic crack detection. The biaxial stress field in bore holes with nonhomogeneous and anisotropic rock conditions can be defined. Finally, recommendations are given for the development of a testing apparatus utilizing thin-film piezoelectric substrates to detect ultrasonic shear waves capable of underground biaxial measurements.

Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with genes that could encode lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes: β-glucosidase, α-L-arabinofuranosidase, β-xylosidase, and endo-1,4-β-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation. Phylogenetic trees were created for each of these putative enzymes to depict genetic relatedness to known enzymes. Candidate genes were synthesized and cloned into plasmid expression vectors for inducible protein expression and secretion. The putative β-glucosidase fusion protein was efficiently secreted but did not permit Escherichia coli (E. coli) to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl β-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). The putative hemicellulose-degrading β-xylosidase and α-L-arabinofuranosidase enzymes also lacked in vitro enzyme activity, but the affinity purified endo-1,4-β-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7. At this optimal pH, KM, Vmax, and kcat were determined to be 32.005 ± 4.72 μM, 1.16x10-5 ± 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases.

Host diet influences the diversity and metabolic activities of the gut microbiome. Previous studies have shown that the gut microbiome provides a wide array of enzymes that enable processing of diverse dietary components. Because the primary diet of the porcupine, Erethizon dorsatum, is lignified plant material, we reasoned that the porcupine microbiome would be replete with enzymes required to degrade lignocellulose. Here, we report on the bacterial composition in the porcupine microbiome using 16S rRNA sequencing and bioinformatics analysis. We extended this analysis to the microbiomes of 20 additional mammals located in Shubenacadie Wildlife Park (Nova Scotia, Canada), enabling the comparison of bacterial diversity amongst three mammalian taxonomic orders (Rodentia, Carnivora, and Artiodactyla). 16S rRNA sequencing was validated using metagenomic shotgun sequencing on selected herbivores (porcupine, beaver) and carnivores (coyote, Arctic wolf). In the microbiome, functionality is more conserved than bacterial composition, thus we mined microbiome data sets to identify conserved microbial functions across species in each order. We measured the relative gene abundances for cellobiose phosphorylase, endoglucanase, and beta-glucosidase to evaluate the cellulose-degrading potential of select mammals. The porcupine and beaver had higher proportions of genes encoding cellulose-degrading enzymes than the Artic wolf and coyote. These findings provide further evidence that gut microbiome diversity and metabolic capacity are influenced by host diet.

In physiological settings, the complement protein C3 is deposited on all bacteria, including invasive pathogens. However, because experimental host-bacteria systems typically use decomplemented serum to avoid the lytic action of complement, the impact of C3 coating on epithelial cell responses to invasive bacteria remains unexplored. Here, we demonstrate that following invasion, intracellular C3-positive Listeria monocytogenes is targeted by autophagy through a direct C3/ATG16L1 interaction, resulting in autophagy-dependent bacterial growth restriction. In contrast, Shigella flexneri and Salmonella Typhimurium escape autophagy-mediated growth restriction in part through the action of bacterial outer membrane proteases that cleave bound C3. Upon oral infection with Listeria, C3-deficient mice displayed defective clearance at the intestinal mucosa. Together, these results demonstrate an intracellular role of complement in triggering antibacterial autophagy and immunity against intracellular pathogens. Since C3 indiscriminately associates with foreign surfaces, the C3-ATG16L1 interaction may provide a universal mechanism of xenophagy initiation.

Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA.

Shigella species are the aetiological agents of shigellosis, a severe diarrhoeal disease that is a significant cause of morbidity and mortality worldwide. Shigellosis causes massive colonic destruction, high fever and bloody diarrhoea. Shigella pathogenesis is tightly linked to the ability of the bacterium to invade and replicate intracellularly within the colonic epithelium. Shigella uses a type 3 secretion system to deliver its effector proteins into the cytosol of infected cells. Among the repertoire of Shigella effectors, many are known to target components of the actin cytoskeleton to promote bacterial entry. An emerging alternate theme for effector function is the targeting of the host ubiquitin system. Ubiquitination is a post-translational modification restricted to eukaryotes and is involved in many essential host processes. By virtue of sheer number of ubiquitin-modulating effector proteins, it is clear that Shigella has invested heavily into subversion of the ubiquitin system. Understanding these host-pathogen interactions will inform us about the strategies used by successful pathogens and may also provide avenues for novel antimicrobial strategies.

IpaH proteins are bacterium-specific E3 enzymes that function as type three secretion system (T3SS) effectors in Salmonella, Shigella, and other Gram-negative bacteria. IpaH enzymes recruit host substrates for ubiquitination via a leucine-rich repeat (LRR) domain, which can inhibit the catalytic domain in the absence of substrate. The basis for substrate recognition and the alleviation of autoinhibition upon substrate binding is unknown. Here, we report the X-ray structure of Salmonella SspH1 in complex with human PKN1. The LRR domain of SspH1 interacts specifically with the HR1b coiled-coil subdomain of PKN1 in a manner that sterically displaces the catalytic domain from the LRR domain, thereby activating catalytic function. SspH1 catalyzes the ubiquitination and proteasome-dependent degradation of PKN1 in cells, which attenuates androgen receptor responsiveness but not NF-κB activity. These regulatory features are conserved in other IpaH-substrate interactions. Our results explain the mechanism whereby substrate recognition and enzyme autoregulation are coupled in this class of bacterial ubiquitin ligases.

Intestinal epithelial cells (IECs) act as sentinels for incoming pathogens. Cytosol-invasive bacteria, such as Shigella flexneri, trigger a robust pro-inflammatory nuclear factor κB (NF-κB) response from IECs that is believed to depend entirely on the peptidoglycan sensor NOD1. We found that, during Shigella infection, the TRAF-interacting forkhead-associated protein A (TIFA)-dependent cytosolic surveillance pathway, which senses the bacterial metabolite heptose-1,7-bisphosphate (HBP), functions after NOD1 to detect bacteria replicating free in the host cytosol. Whereas NOD1 mediated a transient burst of NF-κB activation during bacterial entry, TIFA sensed HBP released during bacterial replication, assembling into large signaling complexes to drive a dynamic inflammatory response that reflected the rate of intracellular bacterial proliferation. Strikingly, IECs lacking TIFA were unable to discriminate between proliferating and stagnant intracellular bacteria, despite the NOD1/2 pathways being intact. Our results define TIFA as a rheostat for intracellular bacterial replication, escalating the immune response to invasive Gram-negative bacteria that exploit the host cytosol for growth.

Wire-faced walls of mechanically stabilized earth (MSE) provide an economical method for building vertical structures for supporting roadways where local topography or high land costs preclude the use of conventional fill slopes. W-beam guardrail systems are often used for shielding high vertical drop-offs associated with MSE walls. For this study, the Midwest Guardrail System (MGS) was modified to decrease the overall width of the MSE wall structure. Dynamic component testing was used to determine the post–soil behavior of steel and wood posts embedded in compacted soil materials used for constructing wire-faced MSE walls as well as to evaluate the effects of sloped terrain and different installation methods. Twenty-six dynamic tests were performed to evaluate the propensity for MSE wall damage, to select post length, and to determine the preferred post material and section. The standard MGS was modified by removing the wood spacer blocks 12 in. (305 mm) deep and by incorporating W-beam backup plates. All other MGS features—including the W6 × 8.5 (W152 × 12.6) steel posts 6 ft (1.8 m) long, rail splices at midspan locations, the 31-in. (787-mm) top-mounting height, and the 75-in. (1,905-mm) post spacing—were maintained. The nonblocked MGS was installed with the posts driven at the slope break point of a 3H:1V fill slope. The modified MGS was successfully crash tested with both 1100C small car and 2270P pickup truck vehicles in accordance with Test Level 3 safety performance guidelines in the Manual for Assessing Safety Hardware. The MSE wall was not damaged during the testing programs. The nonblocked MGS is recommended for use with wire-faced MSE walls when they are placed at the slope break point of a 3H:1V fill slope. The modified MGS reduces the required width of the MSE wall, resulting in decreased construction costs.

IpaH proteins are E3 ubiquitin ligases delivered by the type III secretion apparatus into host cells upon infection of humans by the Gram-negative pathogen Shigella flexneri. These proteins comprise a variable leucine-rich repeat-containing N-terminal domain and a conserved C-terminal domain harboring an invariant cysteine residue that is crucial for activity. IpaH homologs are encoded by diverse animal and plant pathogens. Here we demonstrate that the IpaH C-terminal domain carries the catalytic activity for ubiquitin transfer and that the N-terminal domain carries the substrate specificity. The structure of the IpaH C-terminal domain, determined to 2.65-A resolution, represents an all-helical fold bearing no resemblance to previously defined E3 ubiquitin ligases. The conserved and essential cysteine residue lies on a flexible, surface-exposed loop surrounded by conserved acidic residues, two of which are crucial for IpaH activity.

The yeast Saccharomyces cerevisiae senses and responds to nutrients by adapting its growth rate and undergoing morphogenic transitions to ensure survival. The Tor pathway is a major integrator of nutrient-derived signals that in coordination with other signaling pathways orchestrates cell growth. Recent advances have identified novel Tor kinase substrates and established the protein trafficking membranous network and the nucleus as platforms for Tor signaling. These and other recent findings delineate distinct signaling branches emanating from membrane-associated Tor complexes to control cell growth.

A new trailer truck-mounted attenuator protection system was designed and successfully crash tested according to the safety performance criteria presented in NCHRP Report 350. The new trailer is designed to be attached to a tow vehicle using a conventional pintle hitch and wiring harness. This simple attachment will allow the new trailer to be attached directly to many work trucks, such as striping vehicles to eliminate the need for secondary shadow vehicles. The trailer is manufactured from heavy gage, galvanized steel to provide the first practical solution to protecting motorists during snow plowing, sanding and salting operations. Five full-scale crash tests were successfully conducted with the trailer attached to a tow vehicle that was blocked against forward movement and rotation to simulate an infinitely heavy vehicle.

This paper presents findings of an investigation of the safety performance of culvert safety grates when installed on slopes as steep as 3:1, as recommended by the AASHTO Roadside Design Guide (RDG). LS-DYNA modeling was used to identify critical impact conditions for roadside culvert grates installed on 3:1 slopes. Two full-scale vehicle crash tests were conducted under the guidelines of NCHRP Report 350 on a 6.4- × 6.4-m (21-×21-ft) culvert safety grate installed on a 3:1 slope. The full-scale crash tests demonstrated that the AASHTO RDG recommended safety grates provide acceptable safety performance when installed on 3:1 slopes.

On the basis of the proposed changes to the NCHRP Report 350 guidelines, NCHRP Project 22-14(2) researchers deemed it appropriate to first evaluate two strong-post W-beam guardrail systems before finalizing the new crash testing procedures and guidelines. For this effort, the modified G4(1S) W-beam guardrail system and the Midwest Guardrail System (MGS) were selected for evaluation and comparison. Five full-scale vehicle crash tests were performed with the two longitudinal barrier systems, in accordance with the Test Level 3 (TL-3) requirements presented in the NCHRP Report 350 Update. For the modified G4(1S) testing program, two 2,270-kg pickup truck vehicles (2270P vehicles) were used: one 3/4-ton, two-door vehicle and one 1/2-ton, four-door vehicle. For the MGS testing program, two 2,270-kg pickup truck vehicles (2270P vehicles) and one 1,100-kg small-car vehicle (an 1100C vehicle) were used, with both pickup truck configurations being evaluated. On the basis of several findings, the NCHRP Project 22-14(2) researchers determined that the 1/2-ton, four-door pickup truck was better suited for use as a surrogate light truck test vehicle than the 3/4-ton, two-door pickup truck. The modified G4(1S) W-beam guardrail system, mounted at the top rail height of 706 mm, provided acceptable safety performance when it was crashed into by the 1/2-ton, four-door pickup truck vehicle, thus meeting the proposed TL-3 requirements presented in the NCHRP Report 350 Update. Testing of the modified G4(1S) W-beam guardrail system was not successful with a 3/4-ton, two-door pickup truck under the TL-3 impact conditions. The MGS was found to meet the TL-3 criteria presented in the NCHRP Report 350 Update for Test Designations 3-10 and 3-11. Satisfactory safety performance was observed with the MGS with both the 1/2-ton, four-door and 3/4-ton, two-door pickup truck vehicles.

Bacterial type III secretion machines are widely used to inject virulence proteins into eukaryotic host cells. These secretion machines are evolutionarily related to bacterial flagella and consist of a large cytoplasmic complex, a transmembrane basal body, and an extracellular needle. The cytoplasmic complex forms a sorting platform essential for effector selection and needle assembly, but it remains largely uncharacterized. Here we use high-throughput cryoelectron tomography (cryo-ET) to visualize intact machines in a virulent Shigella flexneri strain genetically modified to produce minicells capable of interaction with host cells. A high-resolution in situ structure of the intact machine determined by subtomogram averaging reveals the cytoplasmic sorting platform, which consists of a central hub and six spokes, with a pod-like structure at the terminus of each spoke. Molecular modeling of wild-type and mutant machines allowed us to propose a model of the sorting platform in which the hub consists mainly of a hexamer of the Spa47 ATPase, whereas the MxiN protein comprises the spokes and the Spa33 protein forms the pods. Multiple contacts among those components are essential to align the Spa47 ATPase with the central channel of the MxiA protein export gate to form a unique nanomachine. The molecular architecture of the Shigella type III secretion machine and its sorting platform provide the structural foundation for further dissecting the mechanisms underlying type III secretion and pathogenesis and also highlight the major structural distinctions from bacterial flagella.

Shigellosis is a severe diarrheal disease that affects hundreds of thousands of individuals resulting in significant morbidity and mortality worldwide. Shigellosis is caused by Shigella spp., a gram-negative bacterium that uses a Type 3 Secretion System (T3SS) to deliver effector proteins into the cytosol of infected human cells. Shigella infection triggers multiple signaling programs that result in a robust host transcriptional response that includes the induction of multiple proinflammatory cytokines. PML nuclear bodies (PML-NBs) are dynamic subnuclear structures that coordinate immune signaling programs and have a demonstrated role in controlling viral infection. We show that PML-NB number increases upon Shigella infection. We examined the effects of Shigella infection on SUMOylation and found that upon Shigella infection the localization of SUMOylated proteins is altered and the level of SUMOylated proteins decreases. Although Shigella infection does not alter the abundance of SUMO activating enzymes SAE1 or SAE2, it dramatically decreases the level of the SUMO conjugating enzyme Ubc9. All Shigella-induced alterations to the SUMOylation system are dependent upon a T3SS. Thus, we demonstrate that Shigella uses one or more T3SS effectors to influence both PML-NB number and the SUMOylation machinery in human cells.

The Midwest guardrail system (MGS), developed at the Midwest Roadside Safety Facility, was designed to improve the performance of traditional strong-post, W-beam guardrail systems. These improvements include decreasing the potential for rollover with high center-of-gravity vehicles, decreasing the potential for rail rupture at the splice locations, and decreasing the sensitivity of the system to the installation rail height. However, safe guardrail termination options for the MGS must be developed before the system can be implemented on the roadside. Two end terminal designs, the sequential kinking terminal (SKT) and the flared energy-absorbing terminal (FLEAT), were partially redesigned and crash tested in conjunction with the MGS according to NCHRP Report 350 criteria. The new versions of the terminals were named the SKT-MGS and the FLEAT-MGS to designate them for use with the MGS. To evaluate the performance of the terminals with the MGS, a series of four full-scale crash tests was conducted: two redirection tests, NCHRP Report 350 Test Designations 3–34 and 3–35, and two head-on impacts, Test Designations 3–30 and 3–31. The results from the four crash tests were found to meet all relevant safety requirements. The SKT-MGS and FLEAT-MGS end terminals are the first successfully tested end terminals for use with the MGS.

Computer simulation and physical testing of posts and guardrail installations were performed to develop a W-beam guardrail system for installation where bedrock is within the normal embedment depth of the posts. Computer simulation was conducted with BARRIER VII to determine optimal post force-deflection properties and the critical impact point for a full-scale vehicle crash test. Physical testing included dynamic testing of posts in various embedment configurations and one full-scale vehicular crash test, which was performed according to the Test Level 3 criteria specified in NCHRP Report 350. Steel W152 X 13.4 (W6 X 9) posts were embedded into an elongated hole drilled into rock and backfilled with ASTM C33 coarse aggregate, size No. 57. The elongated hole was created by drilling three holes 203 mm (8 in.) in diameter on 165-mm (6.5-in.) centers. The full-scale crash test, which was performed with a 2,000-kg pickup truck, was used to verify the safety performance of the design for situations where bedrock is encountered on the surface of the ground. Guidelines based on dynamic post testing were developed for use when the bedrock is encountered at various depths below the surface.

In 1993, FHWA adopted a policy that requires all roadside features placed within the clear zone to be evaluated according to the guidelines contained in NCHRP Report 350. Variable message sign trailers and arrow board trailers have never been tested under these safety evaluation guidelines. A full-scale crash test and a review of work zone accident studies clearly indicate that these trailers are a serious roadside hazard and they are struck in approximately 15% of all work zone intrusion accidents. An attenuating cushion that safely treats the full range of these trailers was developed. Two full-scale crash tests of the trailer attenuating cushion were conducted to verify that the new designs meet NCHRP Report 350 guidelines. The new safety treatments are believed to be relatively inexpensive to build and easy to deploy. The new systems should reduce the severity of many run-off-road crashes without a major increase in the cost of using variable message signs and arrow boards.

A new box-beam guardrail end terminal, known as the box-beam bursting energy absorbing terminal (BEAT), was designed and shown to meet current United States impact safety standards according to guidelines provided by the National Cooperative Highway Research Program (NCHRP). The BEAT is placed on the end of a box-beam guardrail system. When impacted by an errant vehicle, the head of the BEAT pushes down the box-beam rail causing the rail to expand and burst at the corners at a desired energy absorbing rate. Details of the bursting concept are provided along with descriptions of the new terminal and the performed crash tests. Three crash tests were needed to qualify the BEAT terminal according to Test Level 3 evaluation criteria provided by the NCHRP.

A new box-beam burster energy-absorbing single-sided crash cushion (BEAT-SSCC) was designed and crash tested. This energy-absorbing crash cushion is designed to shield a rigid hazard, such as the end of a concrete safety-shaped barrier. Energy-absorbing capabilities of the BEAT-SSCC are based on the bursting tube technology, similar to that used with the box-beam burster energy-absorbing terminal. Five full-scale vehicle crash tests were conducted to evaluate the impact performance of the BEAT-SSCC in accordance with guidelines set forth in NCHRP Report 350: ( a) Test Designation 3-31—pickup truck head-on test; ( b) Test Designation 3-38—pickup truck critical impact point test (two tests to evaluate two different critical impact points); ( c) Test Designation 3-39—pickup truck reverse direction test at midpoint of crash cushion, and ( d) modified Test Designation 3-39—pickup truck reverse direction test at connection to the concrete barrier. The crash cushion performed as designed, and the BEAT-SSCC meets all evaluation criteria for a Test Level 3 crash cushion set forth in NCHRP Report 350. The BEAT-SSCC is being evaluated by FHWA for approval to be used on the National Highway System.

The design and crash test results of a median barrier version of the Flared Energy-Absorbing Terminal, known as FLEAT-MT, are presented. This energy-absorbing terminal is designed for use with a W-beam, strong-post median barrier. The FLEAT-MT terminal uses two standard FLEAT terminals, one for each of the two W-beam rail elements. The energy-absorbing capability of the FLEAT-MT terminal is based on the sequential kinking concept, similar to that used with the Sequential Kinking Terminal and FLEAT guardrail terminals. Three full-scale vehicle crash tests were conducted to evaluate the impact performance of the FLEAT-MT terminal in accordance with guidelines set forth in NCHRP Report 350: Test 3-35—pickup truck redirection test (Test No. FMT-1), Test 3-31—pickup truck head-on test (Test No. FMT-2), and Test 3-39—pickup truck reverse-direction test (Test No. FMT-3M). The terminal performed as designed. The FLEAT-MT terminal meets all evaluation criteria for a Test Level 3 median barrier terminal set forth in NCHRP Report 350. The FLEAT-MT terminal is being evaluated by FHWA for approval to be used on the National Highway System.

A variety of traffic-controlling devices are used in work zones; some of these are not normally found on the roadside or in the traveled way outside of the work zones. These devices are used to enhance the safety of the work zones by controlling the traffic through these areas. Because of the placement of the traffic control devices, the devices themselves may be potentially hazardous to both workers and errant vehicles. The impact performance of many work-zone traffic control devices is mainly unknown, and to date limited crash testing has been conducted under the criteria of NCHRP Report 350: Recommended Procedures for the Safety Performance Evaluation of Highway Features. The results of full-scale crash testing of flexible panel work-zone sign stands were evaluated and analyzed to quantify the features that successful devices shared, as well as common features of those devices that failed salient safety criteria. Parameters considered included sign base and upright properties, sign height, cross-member properties, and ancillary details. Results pointed to three problematic, fundamental design issues: ( a) combinations of base and upright stiffness and strength that generally lead to significant windshield damage, ( b) cross members that lead to windshield damage in the end-on (90°) impact orientation, and ( c) appurtenances that have an impact on performance. Although there are a significant number of variables that control the performance of a given device, these generalizations offer a basis for the evaluation of the fundamental design elements.

A revised guardrail system has been developed that should provide greatly improved performance for high-center-of-gravity light truck vehicles. The barrier incorporates W-beam guardrail and standard W6×9 steel posts. Primary changes to the design include raising the standard rail height to 635 mm, moving rail splices to midspan between posts, increasing blockout size, and increasing the size of post bolt slots. All of these changes were designed to improve the barrier’s performance with high-center-of-gravity vehicles. One full-scale crash test was conducted to verify that the guardrail would perform adequately with mini-sized automobiles when raised to 660 mm to the center of the rail. This test proved that the barrier can provide satisfactory performance when mounted at heights ranging from 550 mm (standard guardrail height) up to 660 mm. Hence, the new guardrail design provides approximately 110 mm (4.4 in.) of mounting height tolerance. When installed at the nominal mounting height of 635 mm, a 75-mm pavement overlay could be applied to the roadway without requiring adjustments to the barrier’s height.

Summary Interferon exposure boosts cell-autonomous immunity for more efficient pathogen control. But how interferon-enhanced immunity protects the cytosol against bacteria and how professionally cytosol-dwelling bacteria avoid clearance are insufficiently understood. Here we demonstrate that the interferon-induced GTPase family of guanylate-binding proteins (GBPs) coats Shigella flexneri in a hierarchical manner reliant on GBP1. GBPs inhibit actin-dependent motility and cell-to-cell spread of bacteria but are antagonized by IpaH9.8, a bacterial ubiquitin ligase secreted into the host cytosol. IpaH9.8 ubiquitylates GBP1, GBP2, and GBP4 to cause the proteasome-dependent destruction of existing GBP coats. This ubiquitin coating of Shigella favors the pathogen as it liberates bacteria from GBP encapsulation to resume actin-mediated motility and cell-to-cell spread. We conclude that an important function of GBP recruitment to S. flexneri is to prevent the spread of infection to neighboring cells while IpaH9.8 helps bacterial propagation by counteracting GBP-dependent cell-autonomous immunity.

Shigella species are the aetiological agents of shigellosis, a severe diarrhoeal disease that is a significant cause of morbidity and mortality worldwide. Shigellosis causes massive colonic destruction, high fever and bloody diarrhoea. Shigella pathogenesis is tightly linked to the ability of the bacterium to invade and replicate intracellularly within the colonic epithelium. Shigella uses a type 3 secretion system to deliver its effector proteins into the cytosol of infected cells. Among the repertoire of Shigella effectors, many are known to target components of the actin cytoskeleton to promote bacterial entry. An emerging alternate theme for effector function is the targeting of the host ubiquitin system. Ubiquitination is a post-translational modification restricted to eukaryotes and is involved in many essential host processes. By virtue of sheer number of ubiquitin-modulating effector proteins, it is clear that Shigella has invested heavily into subversion of the ubiquitin system. Understanding these host-pathogen interactions will inform us about the strategies used by successful pathogens and may also provide avenues for novel antimicrobial strategies.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.

Rapamycin is an immunosuppressive natural product that inhibits the proliferation of T-cells in response to nutrients and growth factors. Rapamycin binds to the peptidyl-prolyl isomerase FKBP12 and forms protein-drug complexes that inhibit signal transduction by the TOR kinases. The FKBP12 and TOR proteins are conserved from fungi to humans, and in both organisms the TOR signaling pathway plays a role in nutrient sensing. In response to nitrogen sources or amino acids, TOR regulates both transcription and translation, enabling cells to appropriately respond to growth-promoting signals. Rapamycin is having a profound impact on clinical medicine and was approved as an immunosuppressant for transplant recipients in 1999. Ongoing clinical studies address new clinical applications for rapamycin as an antiproliferative drug for chemotherapy and invasive cardiology.

A W-beam guardrail system was developed and successfully crash tested for use on a 2:1 foreslope. The guardrail design was constructed with W-beam rails 2.66 mm thick (12 gauge) totaling 53.34 m in length, and it incorporated a half-post spacing section of 17.15 m. The W-beam rail was supported by 15 W150 × 13.5 steel posts 1829 mm long, spaced 1905 mm on center, and 19 W150 × 13.5 steel posts 2134 mm long, spaced 952.5 mm on center. Routed, 150 × 200 × 360 mm wood spacer blockouts were used to block the rail away from each post. The research study included bogie testing on steel posts placed in sloped fill, computer simulation modeling with BARRIER VII, and one full-scale vehicle crash test, using a 2000-kg pickup truck. The test, impacting at a speed of 100.7 km/h and an angle of 28.5 degrees, was conducted and reported in accordance with the Test Level 3 (TL-3) requirements specified in NCHRP Report 350. The safety performance of the W-beam barrier system was determined to be acceptable according to TL-3 criteria.

The current generation of NCHRP Report 350-compliant guardrail terminals all use wooden breakaway posts in the terminal section. The first two end posts are typically breakaway cable terminal posts inserted into steel foundation tubes that are joined with a ground strut to provide the anchorage capacity. Wood is readily available and inexpensive, but it also has many drawbacks. The quality of the wood and the associated breaking forces vary widely. The strength of a wooden post is affected by many factors, including post size, ring density, knot location and size, cracks and checks, species, and moisture content. Results are presented of an effort to design and develop steel breakaway posts for guardrail terminals. The breakaway steel post system described has been successfully tested for use in a tangent terminal. The post exhibited consistent strength for redirection impacts and failed at very low loads during head-on impacts. The breakaway steel post actually improved the redirective performance of the SKT-350 during a 2020-kg (4,450-lb) pickup truck impact at the beginning of the length of need. These breakaway steel posts have been shown to meet NCHRP Report 350 performance criteria and should provide highway agencies with an alternative where wood posts are unacceptable.

A new flared W-beam guardrail terminal that incorporates energy-absorbing technology has been developed and successfully crash tested to meet the criteria presented in NCHRP Report 350. The terminal, designated FLEAT-350, incorporates an impact head designed to dissipate impact energy by producing a series of plastic hinges in the W-beam as the impact head is pushed down the guardrail. The energy-absorption mechanism allows the flared terminal to absorb large amounts of kinetic energy before the vehicle is allowed to gate through the system. Most components of the new terminal are similar to those incorporated in the SKT-350 terminal. The terminal system is 11.4 m (37.5 ft) long and incorporates a straight flare with a final end offset of between 0.76 m (2.5 ft) and 1.2 m (4 ft). The terminal uses two breakaway posts in foundation tubes and five rough-cut controlled-release terminal posts of 150 by 200 mm (6 by 8 in.). The energy-absorbing design minimizes problems associated with vehicles that penetrate the terminal and strike slopes, ditches, or other hazards behind the guardrail while still traveling at a high rate of speed. Furthermore the new design has fewer components and is less sensitive to post location and installation details than some other flared systems.

A new tangent energy-absorbing W-beam guardrail terminal that meets NCHRP Report 350 criteria has been developed. The terminal, designated the SKT-350, dissipates the energy of an encroaching vehicle by producing a series of plastic hinges in the W-beam as the terminal head is pushed down the guardrail. This energy-absorption concept allows for significantly lower dynamic forces on the encroaching vehicle, reducing the vehicle damage, the weight of the terminal head, the propensity for vehicle yaw and roll after impact, and the chances of buckling in the W-beam section. The energy required to move the head down the rail in this design is optimized for current criteria, but by modifying the bending geometry in the head, the average force to displace the head down the rail can be adjusted from values ranging from 11 to 60 kN (2,500 to 13,500 lb), meaning that the system can be easily modified to meet any future changes in safety performance standards. In addition to these important safety advantages, the terminal incorporates a unique cable anchor bracket that closely resembles a breakaway cable terminal anchor and a novel foundation tube design that facilitates the removal of broken posts during repair. Combining the features of reduced forces and head weight, a simple cable box, and more economical soil tubes allows the system to offer the advantages of both reduced cost and improved performance.

An approach guardrail transition for use with concrete safety shape barriers was developed and crash-tested. The transition was constructed with two nested thrie-beam rails, measuring 2.66 mm thick, and supported by nine W150 x 13.5 steel posts. Post spacings consisted of one at 292 mm, five at 476 mm, and three at 952 mm. Structural tube spacer blockouts were used in the transition system. The system successfully met the Test Level 3 requirements specified in NCHRP Report 350: Recommended Procedures for the Safety Performance Evaluation of Highway Features.

A series of steel posts (W150 X 12.6) and timber posts (15.2 X 20.3 cm) were instrumented with soil pressure transducers to assess the soil-post load response to a 1,388-kg bogie striking the post at 33 km/hr. Soil pressure measurements demonstrated the dramatic effects of soil shear strength and modulus on the responses of both timber and wood posts. The differences in the failure mechanisms between stiff and soft cohesive soils and noncohesive soils are demonstrated by both stress distributions and total stresses measured by the pressure transducers. The measurement system has potential applications for measurement of loads during impact testing of guardrail systems and as a tool for developing more appropriate models of soil behavior during impact loading.

Guardrail-strengthening techniques were assessed by full-scale crash testing according to Service Level 2 conditions of NCHRP Report 230 and by BARRIER VII computer simulation. The Kansas Department of Transportation's standard W-beam with steel posts guardrail was strengthened by nesting the W-beam and by reducing the post spacing. Computer simulations with BARRIER VII were used to assess the various strengthening techniques for guardrails with standard and extended post lengths installed in clay and sand. The soil-post stiffness parameters used in the program were obtained by conducting 21 post impact tests with a 1388-kg bogie striking a post at 33 km/hr. The guardrails constructed with W6 × 8.5 steel posts and 15.2 × 20.3-cm timber posts behaved similarly under all test conditions. The density of the clay has a profound effect on the lateral dynamic deflections. Nesting the W-beam to strengthen the guardrail provides very little benefit, whereas reducing the post spacing by half provides the greatest benefit.