Identification of Small-Molecule Modulators of Diguanylate Cyclase by FRET-Based High-Throughput Screening.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure-activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

Pyomelanin-producing Pseudomonas aeruginosa selected during chronic infections have a large chromosomal deletion which confers resistance to pyocins.

When bacterial lineages make the transition from free-living to permanent association with hosts, they can undergo massive gene losses, for which the selective forces within host tissues are unknown. We identified here melanogenic clinical isolates of Pseudomonas aeruginosa with large chromosomal deletions (66 to 270 kbp) and characterized them to investigate how they were selected. When compared with their wild-type parents, melanogenic mutants (i) exhibited a lower fitness in growth conditions found in human tissues, such as hyperosmolarity and presence of aminoglycoside antibiotics, (ii) narrowed their metabolic spectrum with a growth disadvantage with particular carbon sources, including aromatic amino acids and acyclic terpenes, suggesting a reduction of metabolic flexibility. Despite an impaired fitness in rich media, melanogenic mutants can inhibit their wild-type parents and compete with them in coculture. Surprisingly, melanogenic mutants became highly resistant to two intraspecific toxins, the S-pyocins AP41 and S1. Our results suggest that pyocins produced within a population of infecting P. aeruginosa may have selected for bacterial mutants that underwent massive gene losses and that were adapted to the life in diverse bacterial communities in the human host. Intraspecific interactions may therefore be an important factor driving the continuing evolution of pathogens during host infections.

Pseudomonas aeruginosa in vitro phenotypes distinguish cystic fibrosis infection stages and outcomes.

RATIONALE: Pseudomonas aeruginosa undergoes phenotypic changes during cystic fibrosis (CF) lung infection. Although mucoidy is traditionally associated with transition to chronic infection, we hypothesized that additional in vitro phenotypes correlate with this transition and contribute to disease. OBJECTIVES: To characterize the relationships between in vitro P. aeruginosa phenotypes, infection stage, and clinical outcomes. METHODS: A total of 649 children with CF and newly identified P. aeruginosa were followed for a median 5.4 years during which a total of 2,594 P. aeruginosa isolates were collected. Twenty-six in vitro bacterial phenotypes were assessed among the isolates, including measures of motility, exoproduct production, colony morphology, growth, and metabolism. MEASUREMENTS AND MAIN RESULTS: P. aeruginosa phenotypes present at the time of culture were associated with both stage of infection (new onset, intermittent, or chronic) and the primary clinical outcome, occurrence of a pulmonary exacerbation (PE) in the subsequent 2 years. Two in vitro P. aeruginosa phenotypes best distinguished infection stages: pyoverdine production (31% of new-onset cultures, 48% of intermittent, 69% of chronic) and reduced protease production (31%, 39%, and 65%, respectively). The best P. aeruginosa phenotypic predictors of subsequent occurrence of a PE were mucoidy (odds ratio, 1.75; 95% confidence interval, 1.19-2.57) and reduced twitching motility (odds ratio, 1.43; 95% confidence interval, 1.11-1.84). CONCLUSIONS: In this large epidemiologic study of CF P. aeruginosa adaptation, P. aeruginosa isolates exhibited two in vitro phenotypes that best distinguished early and later infection stages. Among the many phenotypes tested, mucoidy and reduced twitching best predicted subsequent PE. These phenotypes indicate potentially useful prognostic markers of transition to chronic infection and advancing lung disease.

Pseudomonas aeruginosa phenotypes associated with eradication failure in children with cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa is a key respiratory pathogen in people with cystic fibrosis (CF). Due to its association with lung disease progression, initial detection of P. aeruginosa in CF respiratory cultures usually results in antibiotic treatment with the goal of eradication. Pseudomonas aeruginosa exhibits many different phenotypes in vitro that could serve as useful prognostic markers, but the relative relationships between these phenotypes and failure to eradicate P. aeruginosa have not been well characterized. METHODS: We measured 22 easily assayed in vitro phenotypes among the baseline P. aeruginosa isolates collected from 194 participants in the 18-month EPIC clinical trial, which assessed outcomes after antibiotic eradication therapy for newly identified P. aeruginosa. We then evaluated the associations between these baseline isolate phenotypes and subsequent outcomes during the trial, including failure to eradicate after antipseudomonal therapy, emergence of mucoidy, and occurrence of an exacerbation. RESULTS: Baseline P. aeruginosa isolates frequently exhibited phenotypes thought to represent chronic adaptation, including mucoidy. Wrinkly colony surface and irregular colony edges were both associated with increased risk of eradication failure (hazard ratios [95% confidence intervals], 1.99 [1.03-3.83] and 2.14 [1.32-3.47], respectively). Phenotypes reflecting defective quorum sensing were significantly associated with subsequent mucoidy, but no phenotype was significantly associated with subsequent exacerbations during the trial. CONCLUSIONS: Pseudomonas aeruginosa phenotypes commonly considered to reflect chronic adaptation were observed frequently among isolates at early detection. We found that 2 easily assayed colony phenotypes were associated with failure to eradicate after antipseudomonal therapy, both of which have been previously associated with altered biofilm formation and defective quorum sensing.

Evidence for induction of integron-based antibiotic resistance by the SOS response in a clinical setting.

Bacterial resistance to ß-lactams may rely on acquired ß-lactamases encoded by class 1 integron-borne genes. Rearrangement of integron cassette arrays is mediated by the integrase IntI1. It has been previously established that integrase expression can be activated by the SOS response in vitro, leading to speculation that this is an important clinical mechanism of acquiring resistance. Here we report the first in vivo evidence of the impact of SOS response activated by the antibiotic treatment given to a patient and its output in terms of resistance development. We identified a new mechanism of modulation of antibiotic resistance in integrons, based on the insertion of a genetic element, the gcuF1 cassette, upstream of the integron-borne cassette bla(OXA-28) encoding an extended spectrum ß-lactamase. This insertion creates the fused protein GCUF1-OXA-28 and modulates the transcription, the translation, and the secretion of the ß-lactamase in a Pseudomonas aeruginosa isolate (S-Pae) susceptible to the third generation cephalosporin ceftazidime. We found that the metronidazole, not an anti-pseudomonal antibiotic given to the first patient infected with S-Pae, triggered the SOS response that subsequently activated the integrase IntI1 expression. This resulted in the rearrangement of the integron gene cassette array, through excision of the gcuF1 cassette, and the full expression the ß-lactamase in an isolate (R-Pae) highly resistant to ceftazidime, which further spread to other patients within our hospital. Our results demonstrate that in human hosts, the antibiotic-induced SOS response in pathogens could play a pivotal role in adaptation process of the bacteria.

Nutrient availability as a mechanism for selection of antibiotic tolerant Pseudomonas aeruginosa within the CF airway.

Microbes are subjected to selective pressures during chronic infections of host tissues. Pseudomonas aeruginosa isolates with inactivating mutations in the transcriptional regulator LasR are frequently selected within the airways of people with cystic fibrosis (CF), and infection with these isolates has been associated with poorer lung function outcomes. The mechanisms underlying selection for lasR mutation are unknown but have been postulated to involve the abundance of specific nutrients within CF airway secretions. We characterized lasR mutant P. aeruginosa strains and isolates to identify conditions found in CF airways that select for growth of lasR mutants. Relative to wild-type P. aeruginosa, lasR mutants exhibited a dramatic metabolic shift, including decreased oxygen consumption and increased nitrate utilization, that is predicted to confer increased fitness within the nutrient conditions known to occur in CF airways. This metabolic shift exhibited by lasR mutants conferred resistance to two antibiotics used frequently in CF care, tobramycin and ciprofloxacin, even under oxygen-dependent growth conditions, yet selection for these mutants in vitro did not require preceding antibiotic exposure. The selection for loss of LasR function in vivo, and the associated adverse clinical impact, could be due to increased bacterial growth in the oxygen-poor and nitrate-rich CF airway, and from the resulting resistance to therapeutic antibiotics. The metabolic similarities among diverse chronic infection-adapted bacteria suggest a common mode of adaptation and antibiotic resistance during chronic infection that is primarily driven by bacterial metabolic shifts in response to nutrient availability within host tissues.

The bacterial second messenger c-di-GMP: mechanisms of signalling.

Cyclic-di-GMP (c-di-GMP) regulates many important bacterial processes. Freely diffusible intracellular c-di-GMP is determined by the action of metabolizing enzymes that allow integration of numerous input signals. c-di-GMP specifically regulates multiple cellular processes by binding to diverse target molecules. This review highlights important questions in research into the mechanisms of c-di-GMP signalling and its role in bacterial physiology.

Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism.

Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that Pseudomonas aeruginosa possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.

An Ayurvedic Herbal Extract Inhibits Streptococcus mutans Biofilm Formation and Disrupts Preformed Biofilms in vitro.

OBJECTIVE: Sudantha® (SUD), a natural proprietary mixture of herbal extracts that has been incorporated into toothpaste, has been shown in two separate placebo controlled human clinical studies to promote gingival health; and reduce gingival bleeding and plaque formation. However, the herbal based anti-gingivitis mechanisms of Sudantha are not fully understood. The objective of this study was to determine the effect of Sudantha on dental plaque biofilms by investigating its effect on mono-culture biofilms of a primary colonizer, Streptococcus mutans, in vitro. RESULTS: This study found that SUD contributes to the maintenance of oral health through the inhibition of S. mutans biofilm formation. In addition, SUD disrupted preformed S. mutans biofilms after exposure to SUD for 4 hours. Together, this pilot data suggests the inhibition of S. mutans biofilm formation and disruption represents one potential mechanism by which the herbal extract is able to reduce the oral bacterial biofilm resulting in its effective against gingivitis and its potential use in countering biofilm associated oral disease.

Precursor acquisition independent from ion count: how to dive deeper into the proteomics ocean.

Data-dependent precursor ion selection is widely used in shotgun proteomics to profile the protein components of complex samples. Although very popular, this bottom-up method presents major drawbacks in terms of detectable dynamic range. Here, we demonstrate the superior performance of a data-independent method we term precursor acquisition independent from ion count (PAcIFIC). Our results show that almost the entire, predicted, soluble bacterial proteome can be thoroughly analyzed by PAcIFIC without the need for any sample fractionation other than the C18-based liquid chromatograph used to introduce the peptide mixture into the mass spectrometer. Importantly, we also show that PAcIFIC provides unique performance for analysis of human plasma in terms of the number of proteins identified (746 at FDR < or = 0.5%) and achieved dynamic range (8 orders of magnitude at FDR < or = 0.5%), without any fractionation other than immuno-depletion of the seven most abundant proteins.

Pseudomonas aeruginosa lasR mutants are associated with cystic fibrosis lung disease progression.

BACKGROUND: Pseudomonas aeruginosa with mutations in the transcriptional regulator LasR chronically infect the airways of people with cystic fibrosis (CF), yet the prevalence and clinical implications of lasR mutant infection are unknown. METHODS: In an exploratory study, we screened 166 P. aeruginosa isolates from 58 CF patients for LasR inactivation and mucoidy, and compared clinical characteristics among source patients. RESULTS: lasR mutation prevalence was comparable to that of mucoidy, the best-described CF-adapted phenotype, but affected patients were on average approximately 2 years younger. In a regression analysis, lung function decline with age was worse among patients with lasR mutant infection than in those without, similar to the effect of mucoidy. CONCLUSIONS: Culture positivity for lasR mutant P. aeruginosa may serve as a marker of early CF adaptive change of prognostic significance. Furthermore, as LasR inactivation alters susceptibility to antibiotics, infection with lasR mutant P. aeruginosa may impact response to therapy.

The Acinetobacter baumannii Mla system and glycerophospholipid transport to the outer membrane.

The outer membrane (OM) of Gram-negative bacteria serves as a selective permeability barrier that allows entry of essential nutrients while excluding toxic compounds, including antibiotics. The OM is asymmetric and contains an outer leaflet of lipopolysaccharides (LPS) or lipooligosaccharides (LOS) and an inner leaflet of glycerophospholipids (GPL). We screened Acinetobacter baumannii transposon mutants and identified a number of mutants with OM defects, including an ABC transporter system homologous to the Mla system in E. coli. We further show that this opportunistic, antibiotic-resistant pathogen uses this multicomponent protein complex and ATP hydrolysis at the inner membrane to promote GPL export to the OM. The broad conservation of the Mla system in Gram-negative bacteria suggests the system may play a conserved role in OM biogenesis. The importance of the Mla system to Acinetobacter baumannii OM integrity and antibiotic sensitivity suggests that its components may serve as new antimicrobial therapeutic targets.

Conditional toxicity and synergy drive diversity among antibacterial effectors.

Bacteria in polymicrobial habitats contend with a persistent barrage of competitors, often under rapidly changing environmental conditions 1 . The direct antagonism of competitor cells is thus an important bacterial survival strategy 2 . Towards this end, many bacterial species employ an arsenal of antimicrobial effectors with multiple activities; however, the benefits conferred by the simultaneous deployment of diverse toxins are unknown. Here we show that the multiple effectors delivered to competitor bacteria by the type VI secretion system (T6SS) of Pseudomonas aeruginosa display conditional efficacy and act synergistically. One of these effectors, Tse4, is most active in high-salinity environments and synergizes with effectors that degrade the cell wall or inactivate intracellular electron carriers. We find Tse4 synergizes with these disparate mechanisms by forming pores that disrupt the ΔΨ component of the proton motive force. Our results provide evidence that the concomitant delivery of a cocktail of effectors serves as a bet-hedging strategy to promote bacterial competitiveness in the face of unpredictable and variable environmental conditions.

A broadly distributed toxin family mediates contact-dependent antagonism between gram-positive bacteria.

The Firmicutes are a phylum of bacteria that dominate numerous polymicrobial habitats of importance to human health and industry. Although these communities are often densely colonized, a broadly distributed contact-dependent mechanism of interbacterial antagonism utilized by Firmicutes has not been elucidated. Here we show that proteins belonging to the LXG polymorphic toxin family present in Streptococcus intermedius mediate cell contact- and Esx secretion pathway-dependent growth inhibition of diverse Firmicute species. The structure of one such toxin revealed a previously unobserved protein fold that we demonstrate directs the degradation of a uniquely bacterial molecule required for cell wall biosynthesis, lipid II. Consistent with our functional data linking LXG toxins to interbacterial interactions in S. intermedius, we show that LXG genes are prevalent in the human gut microbiome, a polymicrobial community dominated by Firmicutes. We speculate that interbacterial antagonism mediated by LXG toxins plays a critical role in shaping Firmicute-rich bacterial communities.

In vivo protein interaction network analysis reveals porin-localized antibiotic inactivation in Acinetobacter baumannii strain AB5075.

The nosocomial pathogen Acinetobacter baumannii is a frequent cause of hospital-acquired infections worldwide and is a challenge for treatment due to its evolved resistance to antibiotics, including carbapenems. Here, to gain insight on A. baumannii antibiotic resistance mechanisms, we analyse the protein interaction network of a multidrug-resistant A. baumannii clinical strain (AB5075). Using in vivo chemical cross-linking and mass spectrometry, we identify 2,068 non-redundant cross-linked peptide pairs containing 245 intra- and 398 inter-molecular interactions. Outer membrane proteins OmpA and YiaD, and carbapenemase Oxa-23 are hubs of the identified interaction network. Eighteen novel interactors of Oxa-23 are identified. Interactions of Oxa-23 with outer membrane porins OmpA and CarO are verified with co-immunoprecipitation analysis. Furthermore, transposon mutagenesis of oxa-23 or interactors of Oxa-23 demonstrates changes in meropenem or imipenem sensitivity in strain AB5075. These results provide a view of porin-localized antibiotic inactivation and increase understanding of bacterial antibiotic resistance mechanisms.

Transposon mutagenesis.

Transposon-based mutagenesis of bacterial genomes is a powerful method to identify genetic elements that control specific phenotypes. The most frequently used transposon tools in Pseudomonas aeruginosa are based either on Himar1 mariner or Tn5 transposases, both of which have been used to generate nonredundant mutant libraries in P. aeruginosa. Here we present a detailed protocol for using Himar1 mariner-based transposon mutagenesis to create mutant libraries in P. aeruginosa.

c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility.

Individual cell heterogeneity is commonly observed within populations, although its molecular basis is largely unknown. Previously, using FRET-based microscopy, we observed heterogeneity in cellular c-di-GMP levels. In this study, we show that c-di-GMP heterogeneity in Pseudomonas aeruginosa is promoted by a specific phosphodiesterase partitioned after cell division. We found that subcellular localization and reduction of c-di-GMP levels by this phosphodiesterase is dependent on the histidine kinase component of the chemotaxis machinery, CheA, and its phosphorylation state. Therefore, individual cell heterogeneity in c-di-GMP concentrations is regulated by the activity and the asymmetrical inheritance of the chemotaxis organelle after cell division. c-di-GMP heterogeneity results in a diversity of motility behaviors. The generation of diverse intracellular concentrations of c-di-GMP by asymmetric partitioning is likely important to the success and survival of bacterial populations within the environment by allowing a variety of motility behaviors. DOI: http://dx.doi.org/10.7554/eLife.01402.001.

Asymmetrical distribution of the second messenger c-di-GMP upon bacterial cell division.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) regulates cellular motility and the synthesis of organelles and molecules that promote adhesion to a variety of biological and nonbiological surfaces. These properties likely require tight spatial and temporal regulation of c-di-GMP concentration. We have developed genetically encoded fluorescence resonance energy transfer (FRET)-based biosensors to monitor c-di-GMP concentrations within single bacterial cells by microscopy. Fluctuations of c-di-GMP were visualized in diverse Gram-negative bacterial species and observed to be cell cycle dependent. Asymmetrical distribution of c-di-GMP in the progeny correlated with the time of cell division and polarization for Caulobacter crescentus and Pseudomonas aeruginosa. Thus, asymmetrical distribution of c-di-GMP was observed as part of cell division, which may indicate an important regulatory step in extracellular organelle biosynthesis or function.

Genome-specific gas-phase fractionation strategy for improved shotgun proteomic profiling of proteotypic peptides.

Gas-phase fractionation (GPF) is an efficient and straightforward method to increase proteome coverage. In this report, optimal m/z ranges were calculated based on genomic complexity and experimental data. Then, theoretical precursor ion densities were calculated in silico from various organisms' genomes and found to corroborate the empirical selection of m/z ranges based on ion density mapping. According to both calculations, the choice of m/z range for most efficient GPF coverage in the lower m/z range should be very narrow and increase as m/z value increases. Next, a systematic LC-MS/MS analysis was performed to confirm this observation. The behavior of data-dependent precursor ion selection and the origin of the observed variability was investigated under three different scan modes of an LTQ-Orbitrap hybrid mass spectrometer. Finally, GPF combined with data-dependent analysis was compared to a targeted, pseudo-multiple reaction monitoring analysis of proteotypic peptides that should be, based on empirical observation of LC-ESI-MS/MS data, detectable. The result of the latter experiment supported our conclusion that data-dependent analysis using rational gas-phase fractionation was sufficient for comprehensive proteomic analysis of the proteotypic peptides in an unfractionated cell lysate.