Toxin-mediated depletion of NAD and NADP drives persister formation in a human pathogen.

Toxin-antitoxin (TA) systems are widespread in bacteria and implicated in genome stability, virulence, phage defense, and persistence. TA systems have diverse activities and cellular targets, but their physiological roles and regulatory mechanisms are often unclear. Here, we show that the NatR-NatT TA system, which is part of the core genome of the human pathogen Pseudomonas aeruginosa, generates drug-tolerant persisters by specifically depleting nicotinamide dinucleotides. While actively growing P. aeruginosa cells compensate for NatT-mediated NAD+ deficiency by inducing the NAD+ salvage pathway, NAD depletion generates drug-tolerant persisters under nutrient-limited conditions. Our structural and biochemical analyses propose a model for NatT toxin activation and autoregulation and indicate that NatT activity is subject to powerful metabolic feedback control by the NAD+ precursor nicotinamide. Based on the identification of natT gain-of-function alleles in patient isolates and on the observation that NatT increases P. aeruginosa virulence, we postulate that NatT modulates pathogen fitness during infections. These findings pave the way for detailed investigations into how a toxin-antitoxin system can promote pathogen persistence by disrupting essential metabolic pathways.

Combining CRISPRi and metabolomics for functional annotation of compound libraries.

Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli. First, we generated a reference map of metabolic changes from CRISPR interference (CRISPRi) with 352 genes in all major essential biological processes. Next, on the basis of the comparison of genetic changes with 1,342 drug-induced metabolic changes, we made de novo predictions of compound functionality and revealed antibacterials with unconventional modes of action (MoAs). We show that our framework, combining dynamic gene silencing with metabolomics, can be adapted as a general strategy for comprehensive high-throughput analysis of compound functionality from bacteria to human cell lines.

A Novel Genome-Editing Platform for Drug-Resistant Acinetobacter baumannii Reveals an AdeR-Unrelated Tigecycline Resistance Mechanism.

Infections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug-resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug-resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study, we developed a highly efficient and versatile genome-editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profiles. We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline-resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC90 of tigecycline from 25 µg/ml in the parental strains to 3.1 µg/ml in the ΔadeR mutants, indicating its importance in the drug resistance phenotype. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole-genome sequencing revealed loss-of-function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline-resistant clinical isolates. This study highlights the development and application of a powerful genome-editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinically relevant strains.

New iron acquisition system in Bacteroidetes.

Capnocytophaga canimorsus, a dog mouth commensal and a member of the Bacteroidetes phylum, causes rare but often fatal septicemia in humans that have been in contact with a dog. Here, we show that C. canimorsus strains isolated from human infections grow readily in heat-inactivated human serum and that this property depends on a typical polysaccharide utilization locus (PUL), namely, PUL3 in strain Cc5. PUL are a hallmark of Bacteroidetes, and they encode various products, including surface protein complexes that capture and process polysaccharides or glycoproteins. The archetype system is the Bacteroides thetaiotaomicron Sus system, devoted to starch utilization. Unexpectedly, PUL3 conferred the capacity to acquire iron from serotransferrin (STF), and this capacity required each of the seven encoded proteins, indicating that a whole Sus-like machinery is acting as an iron capture system (ICS), a new and unexpected function for Sus-like machinery. No siderophore could be detected in the culture supernatant of C. canimorsus, suggesting that the Sus-like machinery captures iron directly from transferrin, but this could not be formally demonstrated. The seven genes of the ICS were found in the genomes of several opportunistic pathogens from the Capnocytophaga and Prevotella genera, in different isolates of the severe poultry pathogen Riemerella anatipestifer, and in strains of Bacteroides fragilis and Odoribacter splanchnicus isolated from human infections. Thus, this study describes a new type of ICS that evolved in Bacteroidetes from a polysaccharide utilization system and most likely represents an important virulence factor in this group.

Activation and polar sequestration of PopA, a c-di-GMP effector protein involved in Caulobacter crescentus cell cycle control.

When Caulobacter crescentus enters S-phase the replication initiation inhibitor CtrA dynamically positions to the old cell pole to be degraded by the polar ClpXP protease. Polar delivery of CtrA requires PopA and the diguanylate cyclase PleD that positions to the same pole. Here we present evidence that PopA originated through gene duplication from its paralogue response regulator PleD and subsequent co-option as c-di-GMP effector protein. While the C-terminal catalytic domain (GGDEF) of PleD is activated by phosphorylation of the N-terminal receiver domain, functional adaptation has reversed signal transduction in PopA with the GGDEF domain adopting input function and the receiver domain serving as regulatory output. We show that the N-terminal receiver domain of PopA specifically interacts with RcdA, a component required for CtrA degradation. In contrast, the GGDEF domain serves to target PopA to the cell pole in response to c-di-GMP binding. In agreement with the divergent activation and targeting mechanisms, distinct markers sequester PleD and PopA to the old cell pole upon S-phase entry. Together these data indicate that PopA adopted a novel role as topology specificity factor to help recruit components of the CtrA degradation pathway to the protease specific old cell pole of C. crescentus.

The YfiBNR signal transduction mechanism reveals novel targets for the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways.

The genetic adaptation of pathogens in host tissue plays a key role in the establishment of chronic infections. While whole genome sequencing has opened up the analysis of genetic changes occurring during long-term infections, the identification and characterization of adaptive traits is often obscured by a lack of knowledge of the underlying molecular processes. Our research addresses the role of Pseudomonas aeruginosa small colony variant (SCV) morphotypes in long-term infections. In the lungs of cystic fibrosis patients, the appearance of SCVs correlates with a prolonged persistence of infection and poor lung function. Formation of P. aeruginosa SCVs is linked to increased levels of the second messenger c-di-GMP. Our previous work identified the YfiBNR system as a key regulator of the SCV phenotype. The effector of this tripartite signaling module is the membrane bound diguanylate cyclase YfiN. Through a combination of genetic and biochemical analyses we first outline the mechanistic principles of YfiN regulation in detail. In particular, we identify a number of activating mutations in all three components of the Yfi regulatory system. YfiBNR is shown to function via tightly controlled competition between allosteric binding sites on the three Yfi proteins; a novel regulatory mechanism that is apparently widespread among periplasmic signaling systems in bacteria. We then show that during long-term lung infections of CF patients, activating mutations invade the population, driving SCV formation in vivo. The identification of mutational "scars" in the yfi genes of clinical isolates suggests that Yfi activity is both under positive and negative selection in vivo and that continuous adaptation of the c-di-GMP network contributes to the in vivo fitness of P. aeruginosa during chronic lung infections. These experiments uncover an important new principle of in vivo persistence, and identify the c-di-GMP network as a valid target for novel anti-infectives directed against chronic infections.

The lipopolysaccharide from Capnocytophaga canimorsus reveals an unexpected role of the core-oligosaccharide in MD-2 binding.

Capnocytophaga canimorsus is a usual member of dog's mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a "hybrid backbone" lacking the 4' phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 - lipid A complex in case the 4' phosphate is not present.

Pseudomonas aeruginosa breaches respiratory epithelia through goblet cell invasion in a microtissue model.

Pseudomonas aeruginosa, a leading cause of severe hospital-acquired pneumonia, causes infections with up to 50% mortality rates in mechanically ventilated patients. Despite some knowledge of virulence factors involved, it remains unclear how P. aeruginosa disseminates on mucosal surfaces and invades the tissue barrier. Using infection of human respiratory epithelium organoids, here we observed that P. aeruginosa colonization of apical surfaces is promoted by cyclic di-GMP-dependent asymmetric division. Infection with mutant strains revealed that Type 6 Secretion System activities promote preferential invasion of goblet cells. Type 3 Secretion System activity by intracellular bacteria induced goblet cell death and expulsion, leading to epithelial rupture which increased bacterial translocation and dissemination to the basolateral epithelium. These findings show that under physiological conditions, P. aeruginosa uses coordinated activity of a specific combination of virulence factors and behaviours to invade goblet cells and breach the epithelial barrier from within, revealing mechanistic insight into lung infection dynamics.

Phage Paride can kill dormant, antibiotic-tolerant cells of Pseudomonas aeruginosa by direct lytic replication.

Bacteriophages are ubiquitous viral predators that have primarily been studied using fast-growing laboratory cultures of their bacterial hosts. However, microbial life in nature is mostly in a slow- or non-growing, dormant state. Here, we show that diverse phages can infect deep-dormant bacteria and suspend their replication until the host resuscitates ("hibernation"). However, a newly isolated Pseudomonas aeruginosa phage, named Paride, can directly replicate and induce the lysis of deep-dormant hosts. While non-growing bacteria are notoriously tolerant to antibiotic drugs, the combination with Paride enables the carbapenem meropenem to eradicate deep-dormant cultures in vitro and to reduce a resilient bacterial infection of a tissue cage implant in mice. Our work might inspire new treatments for persistent bacterial infections and, more broadly, highlights two viral strategies to infect dormant bacteria (hibernation and direct replication) that will guide future studies on phage-host interactions.

The N-glycan glycoprotein deglycosylation complex (Gpd) from Capnocytophaga canimorsus deglycosylates human IgG.

C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-ß-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including ß-galactosidases, ß-N-Acetylhexosaminidases and α-mannosidases.

The genome and surface proteome of Capnocytophaga canimorsus reveal a key role of glycan foraging systems in host glycoproteins deglycosylation.

Capnocytophaga canimorsus are commensal Gram-negative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria-Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.

Complete genome sequence of the dog commensal and human pathogen Capnocytophaga canimorsus strain 5.

Capnocytophaga canimorsus is a commensal Gram-negative bacterium, originally isolated from a dog's mouth, that causes septicemia in humans. C. canimorsus has the unusual ability to feed on host cells, including phagocytes. This capacity depends on surface-exposed glycan-foraging systems. Here we present the first complete genome sequence of a C. canimorsus strain (Cc5).

The Use of Experimental Evolution to Study the Response of Pseudomonas aeruginosa to Single or Double Antibiotic Treatment.

The widespread use of antibiotics promotes the evolution and dissemination of drug resistance and tolerance. Both mechanisms promote survival during antibiotic exposure and their role and development can be studied in vitro with different assays to document the gradual adaptation through the selective enrichment of resistant or tolerant mutant variants. Here, we describe the use of experimental evolution in combination with time-resolved genome analysis as a powerful tool to study the interaction of antibiotic tolerance and resistance in the human pathogen Pseudomonas aeruginosa . This method guides the identification of components involved in alleviating antibiotic stress and helps to unravel specific molecular pathways leading to drug tolerance or resistance. We discuss the influence of single or double drug treatment regimens and environmental aspects on the evolution of antibiotic resilience mechanisms.

Pareto optimality between growth-rate and lag-time couples metabolic noise to phenotypic heterogeneity in Escherichia coli.

Despite mounting evidence that in clonal bacterial populations, phenotypic variability originates from stochasticity in gene expression, little is known about noise-shaping evolutionary forces and how expression noise translates to phenotypic differences. Here we developed a high-throughput assay that uses a redox-sensitive dye to couple growth of thousands of bacterial colonies to their respiratory activity and show that in Escherichia coli, noisy regulation of lower glycolysis and citric acid cycle is responsible for large variations in respiratory metabolism. We found that these variations are Pareto optimal to maximization of growth rate and minimization of lag time, two objectives competing between fermentative and respiratory metabolism. Metabolome-based analysis revealed the role of respiratory metabolism in preventing the accumulation of toxic intermediates of branched chain amino acid biosynthesis, thereby supporting early onset of cell growth after carbon starvation. We propose that optimal metabolic tradeoffs play a key role in shaping and preserving phenotypic heterogeneity and adaptation to fluctuating environments.

Evolution of Antibiotic Tolerance Shapes Resistance Development in Chronic Pseudomonas aeruginosa Infections.

The widespread use of antibiotics promotes the evolution and dissemination of resistance and tolerance mechanisms. To assess the relevance of tolerance and its implications for resistance development, we used in vitro evolution and analyzed the inpatient microevolution of Pseudomonas aeruginosa, an important human pathogen causing acute and chronic infections. We show that the development of tolerance precedes and promotes the acquisition of resistance in vitro, and we present evidence that similar processes shape antibiotic exposure in human patients. Our data suggest that during chronic infections, P. aeruginosa first acquires moderate drug tolerance before following distinct evolutionary trajectories that lead to high-level multidrug tolerance or to antibiotic resistance. Our studies propose that the development of antibiotic tolerance predisposes bacteria for the acquisition of resistance at early stages of infection and that both mechanisms independently promote bacterial survival during antibiotic treatment at later stages of chronic infections.IMPORTANCE Over the past decades, pan-resistant strains of major bacterial pathogens have emerged and have rendered clinically available antibiotics ineffective, putting at risk many of the major achievements of modern medicine, including surgery, cancer therapy, and organ transplantation. A thorough understanding of processes leading to the development of antibiotic resistance in human patients is thus urgently needed. We show that drug tolerance, the ability of bacteria to survive prolonged exposure to bactericidal antibiotics, rapidly evolves in the opportunistic human pathogen Pseudomonas aeruginosa upon recurrent exposures to antibiotics. Our studies show that tolerance protects P. aeruginosa against different classes of antibiotics and that it generally precedes and promotes resistance development. The rapid evolution of tolerance during treatment regimens may thus act as a strong driving force to accelerate antibiotic resistance development. To successfully counter resistance, diagnostic measures and novel treatment strategies will need to incorporate the important role of antibiotic tolerance.

Defining Proteomic Signatures to Predict Multidrug Persistence in Pseudomonas aeruginosa.

Bacterial persisters are difficult to eradicate because of their ability to survive prolonged exposure to a range of different antibiotics. Because they often represent small subpopulations of otherwise drug-sensitive bacterial populations, studying their physiological state and antibiotic stress response remains challenging. Sorting and enrichment procedures of persister fractions introduce experimental biases limiting the significance of follow-up molecular analyses. In contrast, proteome analysis of entire bacterial populations is highly sensitive and reproducible and can be employed to explore the persistence potential of a given strain or isolate. Here, we summarize methodology to generate proteomic signatures of persistent Pseudomonas aeruginosa isolates with variable fractions of persisters. This includes proteome sample preparation, mass spectrometry analysis, and an adaptable machine learning regression pipeline. We show that this generic method can determine a common proteomic signature of persistence among different P. aeruginosa hyper-persister mutants. We propose that this approach can be used as diagnostic tool to gauge antimicrobial persistence of clinical isolates.

PrhG, a transcriptional regulator responding to growth conditions, is involved in the control of the type III secretion system regulon in Ralstonia solanacearum.

The ability of Ralstonia solanacearum to cause disease in plants depends on its type III secretion system (T3SS). The expression of the T3SS and its effector substrates is coordinately controlled by a regulatory cascade, at the bottom of which is HrpB. Transcription of the hrpB gene is activated by a plant-responsive regulator named HrpG, which is a master regulator of a wide array of pathogenicity functions in R. solanacearum. We have identified in the genome of strain GMI1000 a close paralog of hrpG (83% overall similarity at the protein level) that we have named prhG. Despite this high similarity, the expression pattern of prhG is remarkably different from that of hrpG: prhG expression is activated after growth of bacteria in minimal medium but not in the presence of host cells, while hrpG expression is specifically induced in response to plant cell signals. We provide genetic evidence that prhG is a transcriptional regulator that, like hrpG, controls the expression of hrpB and the hrpB-regulated genes under minimal medium conditions. However, the regulatory functions of prhG and hrpG are distinct: prhG has no influence on hrpB expression when the bacteria are in the presence of plant cells, and transcriptomic profiling analysis of a prhG mutant revealed that the PrhG and HrpG regulons have only one pathogenicity target in common, hrpB. Functional complementation experiments indicated that PrhG and HrpG are individually sufficient to activate hrpB expression in minimal medium. Rather surprisingly, a prhG disruption mutant had little impact on pathogenicity, which may indicate that prhG has a minor role in the activation of T3SS genes when R. solanacearum grows parasitically inside the plant. The cross talk between pathogenicity regulatory proteins and environmental signals described here denotes that an intricate network is at the basis of the bacterial disease program.

A Surface-Induced Asymmetric Program Promotes Tissue Colonization by Pseudomonas aeruginosa.

The opportunistic human pathogen Pseudomonas aeruginosa effectively colonizes host epithelia using pili as primary adhesins. Here we uncover a surface-specific asymmetric virulence program that enhances P. aeruginosa host colonization. We show that when P. aeruginosa encounters surfaces, the concentration of the second messenger c-di-GMP increases within a few seconds. This leads to surface adherence and virulence induction by stimulating pili assembly through activation of the c-di-GMP receptor FimW. Surface-attached bacteria divide asymmetrically to generate a piliated, surface-committed progeny (striker) and a flagellated, motile offspring that leaves the surface to colonize distant sites (spreader). Cell differentiation is driven by a phosphodiesterase that asymmetrically positions to the flagellated pole, thereby maintaining c-di-GMP levels low in the motile offspring. Infection experiments demonstrate that cellular asymmetry strongly boosts infection spread and tissue damage. Thus, P. aeruginosa promotes surface colonization and infection transmission through a cooperative virulence program that we termed Touch-Seed-and-Go.