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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.

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.

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.

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.

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.

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.

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.

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.

Bacteria of the genus Shigella are a major cause of death worldwide (L. von Seidlein et al., PLoS Med. 3:e353, 2006). We sequenced the genome of Shigella flexneri strain M90T Sm (serotype 5a) and compared it to the published genome sequence of S. flexneri strain 8401 (serotype 5b).

Yersinia enterocolitica is a facultative intracellular parasite, displaying the ability to grow saprophytically or invade and persist intracellularly in the mammalian reticuloendothelial system. The transition between such diverse environments requires the co-ordinated regulation of specific sets of genes on both the chromosome and virulence plasmid. Temperature has a profound pleiotropic effect on gene expression and phenotypically promotes alterations in cell morphology, outer-membrane protein synthesis, urease production, lipopolysaccharide synthesis, motility, and synthesis of genes involved in invasion of eukaryotic host cells. By examining thermoregulated flagella biosynthesis, we have determined that motility is repressed at 25 degrees C (permissive temperature) with subinhibitory concentrations of novobiocin. These conditions also induce virulence gene expression suggesting novobiocin addition simulates, at least partially, a high-temperature environment. Furthermore, temperature-shift experiments, using Y. enterocolitica containing pACYC184 as a reporter plasmid, indicate that thermo-induced alterations of DNA supercoiling coincide with temperature-induced phenotypic changes. A class of putative DNA gyrase mutant (novobiocin resistant) likewise demonstrates the 37 degrees C phenotype when cultured at 25 degrees C; it is non-motile, urease negative, calcium growth dependent, and positive for Yop expression. These results support a model implicating DNA topology as a contributing factor of Y. enterocolitica thermoregulation.

Many bacterial pathogens rely on effector proteins to disrupt conserved eukaryotic processes. Despite their fundamental biological importance, it has been difficult to elucidate their mode-of-action using standard bioinformatic, biochemical, or genetic approaches. In recent years, surrogate hosts including the budding yeast Saccharomyces cerevisiae have become increasingly popular to aid the study of effectors. Expression of effectors in yeast can result in phenotypes that may be exploited to elucidate the processes they target, gain insight into their enzymatic function, and understand target-effector relationships. Moreover, chemical genomic approaches in yeast may be used to ascribe functions to these proteins as well as identify lead compounds that may be useful for antimicrobial therapies. The recent successes of the yeast system establish it as a standard tool for the study of bacterial effector proteins.

Interferons (IFNs) are released by mammalian cells upon attack by microbial pathogens, alerting neighboring cells to prepare a defense that includes the activation of so-called IFN-stimulated genes. Although this response nearly always limits viral replication, its role during bacterial infection has not been clear. In some cases, the IFN response accelerates bacteria clearance, but in other cases, it results in a more severe disease ( 1 , 2 ). The latter is true for Listeria monocytogenes , the bacterium that causes a range of human illnesses, from gastroenteritis to fatal meningitis. On page 1319 of this issue, Lebreton et al. ( 3 ) identify a new virulence factor, LntA, secreted by L. monocytogenes , that controls the expression of IFN-stimulated genes. The mechanism allows the bacterium to govern both the induction and repression of the host cell immune response, perhaps to optimize conditions for specific stages of infection or colonization of specific tissues.

A common post-translational modification in eukaryotes is the covalent attachment of ubiquitin, a 76 amino acid protein, to specific proteins. Most commonly, ubiquitin is conjugated to e– amino groups of lysine residues, and in unusual cases it can be conjugated to serine and cysteine residues or the terminal amino group of a protein. This process, referred to as ubiquitination (or ubiquitylation), can result in a variety of outcomes for a protein, depending upon how many Ub molecules are attached, whether a polyubiquitin chain is formed, and the nature of the chain. Monoubiquitination can result in relocalization of proteins, while most polyubiquitin chains (e.g., K48 and K11-linked chains) direct proteins for proteasomal degradation. Linkages of Ub formed using Lys 63 or by end-to-end linkages (also known as Met Ub) [1] are not directed to the proteasome and can mediate protein trafficking, scaffolding of protein complexes, or enzyme activation. Ub chains are also used for targeting invading microbes for clearance via xenophagy [2]. Ubiquitination is carried out by a series of enzymes. First, a ubiquitin activating enzyme (E1) forms a thioester with the Cterminus of Ub. The activated Ub is then transferred to one of many (,40 human) ubiquitin conjugating enzymes (E2s). Finally, the E3 enzymes (perhaps over 500 human E3s) direct the transfer of Ub to specific substrates. In eukaryotic cells there are two general classes of E3 ubiquitin ligases. The HECT (Homologous to E6-AP Carboxyl Terminus) and Ring Between Ring (RBR) domain E3s possess an invariant catalytic Cys residue that accepts Ub from a charged E2 before catalyzing transfer of Ub to substrates. Other E3s contain a Really Interesting New Gene (RING) or RING-like domain (U-box) that recruits a charged E2, as well as a domain that recruits substrates. Ub is then transferred from the E2 to the substrate, with the E3 serving primarily as a scaffold. Some RING E3s are single polypeptides, while the cullinRING Ligases (CRLs) are modular multisubunit complexes [3]. Mammalian CRLs are nucleated by one of seven cullin family members, with a RING domain protein that binds to its Cterminus. The N-terminal region of the cullin binds specific cullin adaptor proteins that engage substrate receptor proteins; the most studied class of substrate receptors are the F-Box proteins. CRLs are subject to an additional level of control by a ubiquitin-like modifier protein, Nedd8. E3 enzymes have two important roles. First, they recognize substrates and position them for ubiquitination. Second, E3s dictate the nature of the Ub linkage(s), which will determine the substrate’s fate. For HECT E3s, the Ub chain type is dictated by the C-terminal lobe of the HECT domain [4]. By contrast, RINGtype E3s direct Ub chain type based upon the charged E2 that they recruit [5]. Successful pathogens use proteins that interfere with host cell function that are delivered into eukaryotic host cells via specialized secretion systems and collectively referred to as ‘‘effectors.’’ Remarkably, although ubiquitin is restricted to eukaryotic cells, the past decade has revealed that both bacterial and viral pathogens use effectors to interfere with or manipulate the ubiquitination system [6]. This involves a large number of Bacterially encoded E3 ubiquitin Ligases (BELs). There are multiple RING-type BELs, HECT-like BELs, and even BELs that bear no resemblance to known eukaryotic ubiquitin ligases (Figure 1A).

Temperature has a pleiotropic effect on Yersinia enterocolitica gene expression. Temperature-dependent phenotypes include the switching between two type III protein secretion systems, flagellum biosynthesis (≤30°C) and virulence plasmid-encoded Yop secretion (37°C). The mechanism by which temperature exerts this change in genetic programming is unclear; however, altered gene expression by temperature-dependent changes in DNA topology has been implicated. Here, we present evidence that the Y. enterocolitica virulence plasmid, pYV, undergoes a conformational transition between 30 and 37°C. Using a simplified two-dimensional, single-gel assay, we show that pYV contains multiple regions of intrinsic curvature, including virF , the positive activator of virulence genes. These bends are detectable at 30°C but melt at 37°C, the temperature at which the cells undergo phenotypic switching. We also show that pACYC184, a plasmid used as a reporter of temperature-induced changes in DNA supercoiling, has a single region of intrinsic bending detected by our assay. Topoisomers of pACYC184, with and without this bend, isolated from Y. enterocolitica were resolved by using chloroquine gels. The single bend has a dramatic influence on temperature-dependent DNA supercoiling. These data suggest that the Y. enterocolitica pYV plasmid may undergo a conformational change at the host temperature due to melting of DNA bends followed by compensatory adjustments in superhelical density. Hence, changes in DNA topology may be the temperature-sensing mechanism for virulence gene expression in Y. enterocolitica and other enteric pathogens.