Distribution and molecular evolution of the anti-CRISPR family AcrIF7.

Anti-clustered regularly interspaced short palindromic repeats (CRISPRs) are proteins capable of blocking CRISPR-Cas systems and typically their genes are located on mobile genetic elements. Since their discovery, numerous anti-CRISPR families have been identified. However, little is known about the distribution and sequence diversity of members within a family, nor how these traits influence the anti-CRISPR's function and evolution. Here, we use AcrIF7 to explore the dissemination and molecular evolution of an anti-CRISPR family. We uncovered 5 subclusters and prevalent anti-CRISPR variants within the group. Remarkably, AcrIF7 homologs display high similarity despite their broad geographical, ecological, and temporal distribution. Although mainly associated with Pseudomonas aeruginosa, AcrIF7 was identified in distinct genetic backgrounds indicating horizontal dissemination, primarily by phages. Using mutagenesis, we recreated variation observed in databases but also extended the sequence diversity of the group. Characterisation of the variants identified residues key for the anti-CRISPR function and other contributing to its mutational tolerance. Moreover, molecular docking revealed that variants with affected function lose key interactions with its CRISPR-Cas target. Analysis of publicly available data and the generated variants suggests that the dominant AcrIF7 variant corresponds to the minimal and optimal anti-CRISPR selected in the family. Our study provides a blueprint to investigate the molecular evolution of anti-CRISPR families.

The Glyoxylate Shunt, 60 Years On.

2017 marks the 60th anniversary of Krebs' seminal paper on the glyoxylate shunt (and coincidentally, also the 80th anniversary of his discovery of the citric acid cycle). Sixty years on, we have witnessed substantial developments in our understanding of how flux is partitioned between the glyoxylate shunt and the oxidative decarboxylation steps of the citric acid cycle. The last decade has shown us that the beautifully elegant textbook mechanism that regulates carbon flux through the shunt in E. coli is an oversimplification of the situation in many other bacteria. The aim of this review is to assess how this new knowledge is impacting our understanding of flux control at the TCA cycle/glyoxylate shunt branch point in a wider range of genera, and to summarize recent findings implicating a role for the glyoxylate shunt in cellular functions other than metabolism.

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate ß-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Global reprogramming of virulence and antibiotic resistance in Pseudomonas aeruginosa by a single nucleotide polymorphism in elongation factor, fusA1.

Clinical isolates of the opportunistic pathogen Pseudomonas aeruginosa from patients with cystic fibrosis (CF) frequently contain mutations in the gene encoding an elongation factor, FusA1. Recent work has shown that fusA1 mutants often display elevated aminoglycoside resistance due to increased expression of the efflux pump, MexXY. However, we wondered whether these mutants might also be affected in other virulence-associated phenotypes. Here, we isolated a spontaneous gentamicin-resistant fusA1 mutant (FusA1P443L) in which mexXY expression was increased. Proteomic and transcriptomic analyses revealed that the fusA1 mutant also exhibited discrete changes in the expression of key pathogenicity-associated genes. Most notably, the fusA1 mutant displayed greatly increased expression of the Type III secretion system (T3SS), widely considered to be the most potent virulence factor in the P. aeruginosa arsenal, and also elevated expression of the Type VI (T6) secretion machinery. This was unexpected because expression of the T3SS is usually reciprocally coordinated with T6 secretion system expression. The fusA1 mutant also displayed elevated exopolysaccharide production, dysregulated siderophore production, elevated ribosome synthesis, and transcriptomic signatures indicative of translational stress. Each of these phenotypes (and almost all of the transcriptomic and proteomic changes associated with the fusA1 mutation) were restored to levels comparable with that in the progenitor strain by expression of the WT fusA1 gene in trans, indicating that the mutant gene is recessive. Our data show that in addition to elevating antibiotic resistance through mexXY expression (and also additional contributory resistance mechanisms), mutations in fusA1 can lead to highly selective dysregulation of virulence gene expression.

The methylation-independent mismatch repair machinery in Pseudomonas aeruginosa.

Over the last 70 years, we've all gotten used to an Escherichia coli-centric view of the microbial world. However, genomics, as well as the development of improved tools for genetic manipulation in other species, is showing us that other bugs do things differently, and that we cannot simply extrapolate from E. coli to everything else. A particularly good example of this is encountered when considering the mechanism(s) involved in DNA mismatch repair by the opportunistic human pathogen, Pseudomonas aeruginosa (PA). This is a particularly relevant phenotype to examine in PA, since defects in the mismatch repair (MMR) machinery often give rise to the property of hypermutability. This, in turn, is linked with the vertical acquisition of important pathoadaptive traits in the organism, such as antimicrobial resistance. But it turns out that PA lacks some key genes associated with MMR in E. coli, and a closer inspection of what is known (or can be inferred) about the MMR enzymology reveals profound differences compared with other, well-characterized organisms. Here, we review these differences and comment on their biological implications.

Evolutionary plasticity in the allosteric regulator-binding site of pyruvate kinase isoform PykA from Pseudomonas aeruginosa.

Unlike many other well-characterized bacteria, the opportunistic human pathogen Pseudomonas aeruginosa relies exclusively on the Entner-Doudoroff pathway (EDP) for glycolysis. Pyruvate kinase (PK) is the main "pacemaker" of the EDP, and its activity is also relevant for P. aeruginosa virulence. Two distinct isozymes of bacterial PK have been recognized, PykA and PykF. Here, using growth and expression analyses of relevant PK mutants, we show that PykA is the dominant isoform in P. aeruginosa Enzyme kinetics assays revealed that PykA displays potent K-type allosteric activation by glucose 6-phosphate and by intermediates from the pentose phosphate pathway. Unexpectedly, the X-ray structure of PykA at 2.4 Å resolution revealed that glucose 6-phosphate binds in a pocket that is distinct from the binding site reported for this metabolite in the PK from Mycobacterium tuberculosis (the only other available bacterial PK structure containing bound glucose 6-phosphate). We propose a mechanism by which glucose 6-phosphate binding at the allosteric site communicates with the PykA active site. Taken together, our findings indicate remarkable evolutionary plasticity in the mechanism(s) by which PK senses and responds to allosteric signals.

The whole-genome sequence analysis of Enterobacter cloacae strain Ghats1: insights into endophytic lifestyle-associated genomic adaptations.

Enterobacter cloacae is normally considered to be an opportunistic human pathogen. Here, we report on the whole-genome sequence of an endophytic E. cloacae, strain "Ghats1", isolated from leaves of the medicinal plant Coscinium fenestratum Gaertn. Functional analysis of the Ghats1 genome revealed an enrichment for genes involved in the uptake and exchange of nutrients, for chemotaxis and for plant colonization. Unexpectedly though, there were no ORFs belonging to the "virulence factors and antibiotic resistance". Moreover, the presence of hydrolytic enzymes and motility functions reveals the characteristics of an endophyte lifestyle of a bacterium that can colonize and adapt to plant environment. These results provide a better understanding of an endophytic lifestyle through plant-microbe interaction, which can be further exploited as a biocontrol agent.

2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

Gluconeogenic precursor availability regulates flux through the glyoxylate shunt in Pseudomonas aeruginosa.

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to the availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle-glyoxylate shunt branch point in P. aeruginosa has a complex enzymology that is profoundly different from those in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios.

Loving the poison: the methylcitrate cycle and bacterial pathogenesis.

Propionate is an abundant catabolite in nature and represents a rich potential source of carbon for the organisms that can utilize it. However, propionate and propionate-derived catabolites are also toxic to cells, so propionate catabolism can alternatively be viewed as a detoxification mechanism. In this review, we summarize recent progress made in understanding how prokaryotes catabolize propionic acid, how these pathways are regulated and how they might be exploited to develop novel antibacterial interventions.

Strategies for managing rival bacterial communities: Lessons from burying beetles.

The role of bacteria in animal development, ecology and evolution is increasingly well understood, yet little is known of how animal behaviour affects bacterial communities. Animals that benefit from defending a key resource from microbial competitors are likely to evolve behaviours to control or manipulate the animal's associated external microbiota. We describe four possible mechanisms by which animals could gain a competitive edge by disrupting a rival bacterial community: "weeding," "seeding," "replanting" and "preserving." By combining detailed behavioural observations with molecular and bioinformatic analyses, we then test which of these mechanisms best explains how burying beetles, Nicrophorus vespilloides, manipulate the bacterial communities on their carcass breeding resource. Burying beetles are a suitable species to study how animals manage external microbiota because reproduction revolves around a small vertebrate carcass. Parents shave a carcass and apply antimicrobial exudates on its surface, shaping it into an edible nest for their offspring. We compared bacterial communities in mice carcasses that were either fresh, prepared by beetles or unprepared but buried underground for the same length of time. We also analysed bacterial communities in the burying beetle's gut, during and after breeding, to understand whether beetles could be "seeding" the carcass with particular microbes. We show that burying beetles do not "preserve" the carcass by reducing bacterial load, as is commonly supposed. Instead, our results suggest they "seed" the carcass with bacterial groups which are part of the Nicrophorus core microbiome. They may also "replant" other bacteria from the carcass gut onto the surface of their carrion nest. Both these processes may lead to the observed increase in bacterial load on the carcass surface in the presence of beetles. Beetles may also "weed" the bacterial community by eliminating some groups of bacteria on the carcass, perhaps through the production of antimicrobials themselves. Whether these alterations to the bacterial community are adaptive from the beetle's perspective, or are simply a by-product of the way in which the beetles prepare the carcass for reproduction, remains to be determined in future work. In general, our work suggests that animals might use more sophisticated techniques for attacking and disrupting rival microbial communities than is currently appreciated.

Discovery of novel fragments inhibiting O-acetylserine sulphhydrylase by combining scaffold hopping and ligand-based drug design.

Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine biosynthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for further development. This novel chemical tool, endowed with favourable pharmacological characteristics, was successfully developed, and a preliminary Structure-Activity Relationship investigation was carried out.

Synthesis and biological evaluation of 1,2-disubsubstituted 4-quinolone analogues of Pseudonocardia sp. natural products.

A series of analogues of Pseudonocardia sp. natural products were synthesized, which have been reported to possess potent antibacterial activity against Helicobacter pylori and induce growth defects in Escherichia coli and Staphylococcus aureus. Taking inspiration from a methodology used in our total synthesis of natural products, we applied this methodology to access analogues possessing bulky N-substituents, traditionally considered to be challenging scaffolds. Screening of the library provided valuable insights into the structure-activity relationship of the bacterial growth defects, and suggested that selectivity between bacterial species should be attainable. Furthermore, a structurally related series of analogues was observed to inhibit production of the virulence factor pyocyanin in the human pathogen Pseudomonas aeruginosa, which may be a result of their similarity to the Pseudomonas quinolone signal (PQS) quorum sensing autoinducer. This provided new insights regarding the effect of N-substitution in PQS analogues, which has been hitherto underexplored.

Structural and Functional Characterization of Malate Synthase G from Opportunistic Pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen recognized as a critical threat by the World Health Organization because of the dwindling number of effective therapies available to treat infections. Over the past decade, it has become apparent that the glyoxylate shunt plays a vital role in sustaining P. aeruginosa during infection scenarios. The glyoxylate shunt comprises two enzymes: isocitrate lyase and malate synthase isoform G. Inactivation of these enzymes has been reported to abolish the ability of P. aeruginosa to establish infection in a mammalian model system, yet we still lack the structural information to support drug design efforts. In this work, we describe the first X-ray crystal structure of P. aeruginosa malate synthase G in the apo form at 1.62 Å resolution. The enzyme is a monomer composed of four domains and is highly conserved with homologues found in other clinically relevant microorganisms. It is also dependent on Mg2+ for catalysis. Metal ion binding led to a change in the intrinsic fluorescence of the protein, allowing us to quantitate its affinity for Mg2+. We also identified putative drug binding sites in malate synthase G using computational analysis and, because of the high resolution of the experimental data, were further able to characterize its hydration properties. Our data reveal two promising binding pockets in malate synthase G that may be exploited for drug design.

Multifunctional supramolecular polymer networks as next-generation consolidants for archaeological wood conservation.

The preservation of our cultural heritage is of great importance to future generations. Despite this, significant problems have arisen with the conservation of waterlogged wooden artifacts. Three major issues facing conservators are structural instability on drying, biological degradation, and chemical degradation on account of Fe(3+)-catalyzed production of sulfuric and oxalic acid in the waterlogged timbers. Currently, no conservation treatment exists that effectively addresses all three issues simultaneously. A new conservation treatment is reported here based on a supramolecular polymer network constructed from natural polymers with dynamic cross-linking formed by a combination of both host-guest complexation and a strong siderophore pendant from a polymer backbone. Consequently, the proposed consolidant has the ability to chelate and trap iron while enhancing structural stability. The incorporation of antibacterial moieties through a dynamic covalent linkage into the network provides the material with improved biological resistance. Exploiting an environmentally compatible natural material with completely reversible chemistries is a safer, greener alternative to current strategies and may extend the lifetime of many culturally relevant waterlogged artifacts around the world.

Discovery of an inhibitor of the production of the Pseudomonas aeruginosa virulence factor pyocyanin in wild-type cells.

Pyocyanin is a small molecule produced by Pseudomonas aeruginosa that plays a crucial role in the pathogenesis of infections by this notorious opportunistic pathogen. The inhibition of pyocyanin production has been identified as an attractive antivirulence strategy for the treatment of P. aeruginosa infections. Herein, we report the discovery of an inhibitor of pyocyanin production in cultures of wild-type P. aeruginosa which is based around a 4-alkylquinolin-2(1H)-one scaffold. To the best of our knowledge, this is the first reported example of pyocyanin inhibition by a compound based around this molecular framework. The compound may therefore be representative of a new structural sub-class of pyocyanin inhibitors, which could potentially be exploited in in a therapeutic context for the development of critically needed new antipseudomonal agents. In this context, the use of wild-type cells in this study is notable, since the data obtained are of direct relevance to native situations. The compound could also be of value in better elucidating the role of pyocyanin in P. aeruginosa infections. Evidence suggests that the active compound reduces the level of pyocyanin production by inhibiting the cell-cell signalling mechanism known as quorum sensing. This could have interesting implications; quorum sensing regulates a range of additional elements associated with the pathogenicity of P. aeruginosa and there is a wide range of other potential applications where the inhibition of quorum sensing is desirable.

Quorum Sensing Is Accompanied by Global Metabolic Changes in the Opportunistic Human Pathogen Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa uses N-acyl-homoserine lactone (AHL)-dependent quorum sensing (QS) systems to control the expression of secreted effectors. These effectors can be crucial to the ecological fitness of the bacterium, playing roles in nutrient acquisition, microbial competition, and virulence. In this study, we investigated the metabolic consequences of AHL-dependent QS by monitoring the metabolic profile(s) of a lasI rhlI double mutant (unable to make QS signaling molecules) and its wild-type progenitor as they progressed through the growth curve. Analysis of culture supernatants by (1)H-nuclear magnetic resonance ((1)H-NMR) spectroscopy revealed that at the point where AHL concentrations peaked in the wild type, the metabolic footprints (i.e., extracellular metabolites) of the wild-type and lasI rhlI mutant diverged. Subsequent gas chromatography-mass spectrometry (GC-MS)-based analysis of the intracellular metabolome revealed QS-dependent perturbations in around one-third of all identified metabolites, including altered concentrations of tricarboxylic acid (TCA) cycle intermediates, amino acids, and fatty acids. Further targeted fatty acid methyl ester (FAME) GC-MS-based profiling of the cellular total fatty acid pools revealed that QS leads to changes associated with decreased membrane fluidity and higher chemical stability. However, not all of the changes we observed were necessarily a direct consequence of QS; liquid chromatography (LC)-MS analyses revealed that polyamine levels were elevated in the lasI rhlI mutant, perhaps a response to the absence of QS-dependent adaptations. Our data suggest that QS leads to a global readjustment in central metabolism and provide new insight into the metabolic changes associated with QS during stationary-phase adaptation. IMPORTANCE: Quorum sensing (QS) is a transcriptional regulatory mechanism that allows bacteria to coordinate their gene expression profile with the population cell density. The opportunistic human pathogen Pseudomonas aeruginosa uses QS to control the production of secreted virulence factors. In this study, we show that QS elicits a global "metabolic rewiring" in P. aeruginosa. This metabolic rerouting of fluxes is consistent with a variety of drivers, ranging from altered QS-dependent transcription of "metabolic genes" through to the effect(s) of global "metabolic readjustment" as a consequence of QS-dependent exoproduct synthesis, as well as a general stress response, among others. To our knowledge, this is the first study of its kind to assess the global impact of QS on the metabolome.

Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa.

In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors.