Highly Potent Cationic Chitosan Derivatives Coupled to Antimicrobial Peptide Dendrimers to Combat Pseudomonas aeruginosa.

The burden of bacterial wound infections has considerably increased due to antibiotic resistance to most of the currently available antimicrobial drugs. Herein, for the first time, a chemical coupling of two cationic N-aryl (pyridyl and aminocinnamyl) chitosan derivatives to antimicrobial peptide dendrimers (AMPDs) of different generations (first, second, and third) via thioether-haloacetyl reaction is reported. The new chitosan-AMPD conjugates show high selectivity by killing Pseudomonas aeruginosa and very low toxicity toward mammalian cells, as well as extremely low hemolysis to red blood cells. Electron microscopy reveals that the new chitosan derivatives coupled to AMPD destroy both the inner and outer membranes of Gram-negative P. aeruginosa. Moreover, chitosan-AMPD conjugates show synergetic effects within extremely low concentrations. The new chitosan-AMPD conjugates can be used as potent antimicrobial therapeutic agents, to eradicate pathogens such as those present in acute and chronic infected wounds.

Strontium-90 pollution can be bioremediated with the green microalga Tetraselmis chui.

Strontium-90 (90Sr) is an artificial radioisotope produced by nuclear fission, with a relatively long half-life of 29 years. This radionuclide is released into the environment in the event of a nuclear incident, posing a serious risk to human and ecosystem health. There is a need to develop new efficient methods for the remediation of 90Sr, as current techniques for its removal have significant technical limitations and involve high energy and economic costs. Recently, several species of green microalgae within the class Chlorodendrophyceae have been found to form intracellular mineral inclusions of amorphous calcium carbonate (ACC), which can be highly enriched in natural (non-radiogenic) Sr. As bioremediation techniques are an attractive option to address radioactive pollution, we investigated the capacity of the unicellular alga Tetraselmis chui (class Chlorodendrophyceae) to sequester 90Sr. The 90Sr uptake capacity of T. chui cells was assessed in laboratory cultures by monitoring the time course of radioactivity in the culture medium using liquid scintillation counting (LSC). T. chui was shown to effectively sequester 90Sr, reducing the initial radioactivity of the culture medium by up to 50%. Thus, this study demonstrates the potential of the microalga T. chui to be used as a bioremediation agent against 90Sr pollution.

One step bio-guided isolation of secondary metabolites from the endophytic fungus Penicillium crustosum using high-resolution semi-preparative HPLC.

BACKGROUND: An endophytic fungal strain Penicillium crustosum was isolated from the seagrass Posidonia oceanica and investigated to identify its antimicrobial constituents and characterize its metabolome composition. The ethyl acetate extract of this fungus exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) as well as an anti-quorum sensing effect against Pseudomonas aeruginosa. METHODS: The crude extract was profiled by UHPLC-HRMS/MS and the dereplication was assisted by feature-based molecular networking. As a result, more than twenty compounds were annotated in this fungus. To rapidly identify the active compounds, the enriched extract was fractionated by semi-preparative HPLC-UV applying a chromatographic gradient transfer and dry load sample introduction to maximise resolution. The collected fractions were profiled by 1H-NMR and UHPLC-HRMS. RESULTS: The use of molecular networking-assisted UHPLC-HRMS/MS dereplication allowed preliminary identification of over 20 compounds present in the ethyl acetate extract of P. crustosum. The chromatographic approach significantly accelerated the isolation of the majority of compounds present in the active extract. The one-step fractionation allowed the isolation and identification of eight compounds (1-8). CONCLUSION: This study led to the unambiguous identification of eight known secondary metabolites as well as the determination of their antibacterial properties.

A Zur-mediated transcriptional regulation of the zinc export system in Pseudomonas aeruginosa.

The control of cellular zinc (Zn) concentrations by dedicated import and export systems is essential for the survival and virulence of Pseudomonas aeruginosa. The transcription of its many Zn transporters is therefore tightly regulated by a known set of transcription factors involved in either the import or the export of Zn. In this work, we show that the Zur protein, a well-known repressor of Zn import, plays a dual role and functions in both import and export processes. In a situation of Zn excess, Zur represses Zn entry, but also activates the transcription of czcR, a positive regulator of the Zn export system. To achieve this, Zur binds at two sites, located by DNA footprinting in the region downstream the czcR transcription start site. In agreement with this regulation, a delay in induction of the efflux system is observed in the absence of Zur and Zn resistance is reduced. The discovery of this regulation highlights a new role of Zur as global regulator of Zn homeostasis in P. aeruginosa disclosing an important link between Zur and zinc export.

Zinc homeostasis in Pseudomonas.

In the genus Pseudomonas, zinc homeostasis is mediated by a complete set of import and export systems, whose expression is precisely controlled by three transcriptional regulators: Zur, CzcR and CadR. In this review, we describe in detail our current knowledge of these systems, their regulation, and the biological significance of zinc homeostasis, taking Pseudomonas aeruginosa as our paradigm. Moreover, significant parts of this overview are dedicated to highlight interactions and cross-regulations between zinc and copper import/export systems, and to shed light, through a review of the literature and comparative genomics, on differences in gene complement and function across the whole Pseudomonas genus. The impact and importance of zinc homeostasis in Pseudomonas and beyond will be discussed throughout this review.

High calcium and strontium uptake by the green microalga Tetraselmis chui is related to micropearl formation and cell growth.

Strontium-rich micropearls (intracellular inclusions of amorphous calcium carbonate) have been observed in several species of green microalgae within the class Chlorodendrophyceae, suggesting the potential use of these organisms for 90 Sr bioremediation purposes. However, very little is known about the micropearl formation process and the Ca and Sr uptake dynamics of these microalgae. To better understand this phenomenon, we investigated, through laboratory cultures, the behaviour of two species within the class Chorodendrophyceae: Tetraselmis chui, forming micropearls, and T. marina, not forming micropearls. We show that T. chui growth and micropearl formation requires available Ca in the culture medium, and that the addition of dissolved Sr can partially replace the function of Ca in cells. On the other hand, T. marina can grow without added Ca and Sr, probably due to its inability to form micropearls. T. chui cells show a high Ca and Sr uptake, significantly decreasing the concentration of both elements in the culture medium. Strontium is incorporated in micropearls in a short period of time, suggesting that micropearl formation is, most likely, a fast process that only takes a few hours. In addition, we show that micropearls equally distribute between daughter cells during cell division.

Preventing Antibiotic-Resistant Infections: Additively Manufactured Porous Ti6Al4V Biofunctionalized with Ag and Fe Nanoparticles.

Implant-associated infections are highly challenging to treat, particularly with the emergence of multidrug-resistant microbials. Effective preventive action is desired to be at the implant site. Surface biofunctionalization of implants through Ag-doping has demonstrated potent antibacterial results. However, it may adversely affect bone regeneration at high doses. Benefiting from the potential synergistic effects, combining Ag with other antibacterial agents can substantially decrease the required Ag concentration. To date, no study has been performed on immobilizing both Ag and Fe nanoparticles (NPs) on the surface of additively manufactured porous titanium. We additively manufactured porous titanium and biofunctionalized its surface with plasma electrolytic oxidation using a Ca/P-based electrolyte containing Fe NPs, Ag NPs, and the combinations. The specimen's surface morphology featured porous TiO2 bearing Ag and Fe NPs. During immersion, Ag and Fe ions were released for up to 28 days. Antibacterial assays against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa showed that the specimens containing Ag NPs and Ag/Fe NPs exhibit bactericidal activity. The Ag and Fe NPs worked synergistically, even when Ag was reduced by up to three times. The biofunctionalized scaffold reduced Ag and Fe NPs, improving preosteoblasts proliferation and Ca-sensing receptor activation. In conclusion, surface biofunctionalization of porous titanium with Ag and Fe NPs is a promising strategy to prevent implant-associated infections and allow bone regeneration and, therefore, should be developed for clinical application.

Role of Two-Component System Networks in Pseudomonas aeruginosa Pathogenesis.

Two-component systems (TCS) are the largest family of signaling systems in the bacterial kingdom. They enable bacteria to cope with a wide range of environmental conditions via the sensing of stimuli and the transduction of the signal into an appropriate cellular adaptation response. Pseudomonas aeruginosa possesses one of the richest arrays of TCSs in bacteria and they have been the subject of intense investigation for more than 20 years. Most of the P. aeruginosa TCSs characterized to date affect its pathogenesis, via the regulation of virulence factors expression, modulation of the synthesis of antibiotic/antimicrobial resistance mechanisms, and/or via linking virulence to energy metabolism. Here, we give an overview of the current knowledge on P. aeruginosa TCSs, citing key examples for each of the above-mentioned regulatory actions. We then conclude by mentioning few small molecule inhibitors of P. aeruginosa TCSs that have shown an antimicrobial action in vitro.

Chiral Separation of Stilbene Dimers Generated by Biotransformation for Absolute Configuration Determination and Antibacterial Evaluation.

A series of complex stilbene dimers have been generated through biotransformation of resveratrol, pterostilbene, and the mixture of both using the enzymatic secretome of Botrytis cinerea Pers. The process starts with achiral molecules and results in the generation of complex molecules with multiple chiral carbons. So far, we have been studying these compounds in the form of enantiomeric mixtures. In the present study, we isolated the enantiomers to determine their absolute configuration and assess if the stereochemistry could impact their biological properties. Eight compounds were selected for this study, corresponding to the main scaffolds generated (pallidol, leachianol, restrytisol and acyclic dimers) and the most active compounds (trans-δ-viniferin derivatives) against a methicillin-resistant strain of Staphylococcus aureus (MRSA). To isolate these enantiomers and determine their absolute configuration, a chiral HPLC-PDA analysis was performed. The analysis was achieved on a high-performance liquid chromatography system equipped with a chiral column. For each compound, the corresponding enantiomeric pair was obtained with high purity. The absolute configuration of each enantiomer was determined by comparison of experimental and calculated electronic circular dichroism (ECD). The antibacterial activities of the four trans-δ-viniferin derivatives against two S. aureus strains were evaluated.

Synergistic effects of antimicrobial peptide dendrimer-chitosan polymer conjugates against Pseudomonas aeruginosa.

We report herein a new chemical platform for coupling chitosan derivatives to antimicrobial peptide dendrimers (AMPDs) with different degrees of ramification and molecular weights via thiol-maleimide reactions. Previous studies showed that simple incorporation of AMPDs to polymeric hydrogels resulted in a loss of antibacterial activity and augmented cytotoxicity to mammalian cells. We have shown that coupling AMPDs to chitosan derivatives enabled the two compounds to act synergistically. We showed that the antimicrobial activity was preserved when incorporating AMPD conjugates into various biopolymer formulations, including nanoparticles, gels, and foams. Investigating their mechanism of action using electron and time-lapse microscopy, we showed that the AMPD-chitosan conjugates were internalized after damaging outer and inner Gram-negative bacterial membranes. We also showed the absence of AMPD conjugates toxicity to mammalian cells. This chemical technological platform could be used for the development of new membrane disruptive therapeutics to eradicate pathogens present in acute and chronic wounds.

Combination of Pseudo-LC-NMR and HRMS/MS-Based Molecular Networking for the Rapid Identification of Antimicrobial Metabolites From Fusarium petroliphilum.

An endophytic fungal strain isolated from a seagrass endemic to the Mediterranean Sea (Posidonia oceanica) was studied in order to identify its antimicrobial constituents and further characterize the composition of its metabolome. It was identified as Fusarium petroliphilum by in-depth phylogenetic analyses. The ethyl acetate extract of that strain exhibited antimicrobial activities and an ability to inhibit quorum sensing of Staphylococcus aureus. To perform this study with a few tens of mg of extract, an innovative one-step generic strategy was devised. On one side, the extract was analyzed by UHPLC-HRMS/MS molecular networking for dereplication. On the other side, semi-preparative HPLC using a similar gradient profile was used for a single-step high-resolution fractionation. All fractions were systematically profiled by 1H-NMR. The data were assembled into a 2D contour map, which we call "pseudo-LC-NMR," and combined with those of UHPLC-HRMS/MS. This further highlighted the connection within structurally related compounds, facilitated data interpretation, and provided an unbiased quantitative profiling of the main extract constituents. This innovative strategy led to an unambiguous characterization of all major specialized metabolites of that extract and to the localization of its bioactive compounds. Altogether, this approach identified 22 compounds, 13 of them being new natural products and six being inhibitors of the quorum sensing mechanism of S. aureus and Pseudomonas aeruginosa. Minor analogues were also identified by annotation propagation through the corresponding HRMS/MS molecular network, which enabled a consistent annotation of 27 additional metabolites. This approach was designed to be generic and applicable to natural extracts of the same polarity range.

Global Analysis of the Zinc Homeostasis Network in Pseudomonas aeruginosa and Its Gene Expression Dynamics.

Zinc is one of the most important trace elements for life and its deficiency, like its excess, can be fatal. In the bacterial opportunistic pathogen Pseudomonas aeruginosa, Zn homeostasis is not only required for survival, but also for virulence and antibiotic resistance. Thus, the bacterium possesses multiple Zn import/export/storage systems. In this work, we determine the expression dynamics of the entire P. aeruginosa Zn homeostasis network at both transcript and protein levels. Precisely, we followed the switch from a Zn-deficient environment, mimicking the initial immune strategy to counteract bacterial infections, to a Zn-rich environment, representing the phagocyte metal boost used to eliminate an engulfed pathogen. Thanks to the use of the NanoString technology, we timed the global silencing of Zn import systems and the orchestrated induction of Zn export systems. We show that the induction of Zn export systems is hierarchically organized as a function of their impact on Zn homeostasis. Moreover, we identify PA2807 as a novel Zn resistance component in P. aeruginosa and highlight new regulatory links among Zn-homeostasis systems. Altogether, this work unveils a sophisticated and adaptive homeostasis network, which complexity is key in determining a pathogen spread in the environment and during host-colonization.

Characterization of Pseudomonas aeruginosa Quorum Sensing Inhibitors from the Endophyte Lasiodiplodia venezuelensis and Evaluation of Their Antivirulence Effects by Metabolomics.

The opportunistic pathogen Pseudomonas aeruginosa is one of the "critical priority pathogens" due to its multidrug resistance to a wide range of antibiotics. Its ability to invade and damage host tissues is due to the use of quorum sensing (QS) to collectively produce a plethora of virulence factors. Inhibition of QS is an attractive strategy for new antimicrobial agents because it disrupts the initial events of infection without killing the pathogen. Highly diverse microorganisms as endophytes represent an under-explored source of bioactive natural products, offering opportunities for the discovery of novel QS inhibitors (QSI). In the present work, the objective was to explore selective QSIs within a unique collection of fungal endophytes isolated from the tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted, and screened for their antibacterial and specific anti-QS activities against P. aeruginosa. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation for its selective activity, leading to the isolation of eight compounds in a single step. Among them, two pyran-derivatives were found to be responsible for the QSI activity, with an effect on some QS-regulated virulence factors. Additional non-targeted metabolomic studies on P. aeruginosa documented their effects on the production of various virulence-related metabolites.

Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of Lasiodiplodia venezuelensis.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as (R)-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis.

Generation of Stilbene Antimicrobials against Multiresistant Strains of Staphylococcus aureus through Biotransformation by the Enzymatic Secretome of Botrytis cinerea.

The biotransformation of a mixture of resveratrol and pterostilbene was performed by the protein secretome of Botrytis cinerea. Several reaction conditions were tested to overcome solubility issues and to improve enzymatic activity. Using MeOH as cosolvent, a series of unusual methoxylated compounds was generated. The reaction was scaled-up, and the resulting mixture purified by semipreparative HPLC-PDA-ELSD-MS. Using this approach, 15 analogues were isolated in one step. Upon full characterization by NMR and HRMS analyses, eight of the compounds were new. The antibacterial activities of the isolated compounds were evaluated in vitro against the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The selectivity index was calculated based on cytotoxic assays performed against human liver carcinoma cells (HepG2) and the human breast epithelial cell line (MCF10A). Some compounds revealed remarkable antibacterial activity against multidrug-resistant strains of S. aureus with moderate human cell line cytotoxicity.

The CzcCBA Efflux System Requires the CadA P-Type ATPase for Timely Expression Upon Zinc Excess in Pseudomonas aeruginosa.

Zinc (Zn) is a trace element essential for life but can be toxic if present in excess. While cells have import systems to guarantee a vital Zn intracellular concentration, they also rely on export systems to avoid lethal Zn overload. In particular, the opportunistic pathogen Pseudomonas aeruginosa possesses four Zn export systems: CadA, CzcCBA, CzcD, and YiiP. In this work, we compare the importance for bacterial survival of each export system at high Zn concentrations. We show that the P-type ATPase CadA, and the efflux pump CzcCBA are the main efflux systems affecting the bacterium tolerance to Zn. In addition, cadA and czcCBA genes expression kinetics revealed a hierarchical organization and interdependence. In the presence of high Zn concentrations, cadA expression is very rapidly induced (<1 min), while czcCBA expression occurs subsequently (>15 min). Our present data show that the fast responsiveness of cadA to Zn excess is due to its transcriptional activator, CadR, which is constitutively present on its promoter and promptly activating cadA gene expression upon Zn binding. Moreover, we showed that CadA is essential for a timely induction of the CzcCBA efflux system. Finally, we observed an induction of cadA and czcCBA efflux systems upon phagocytosis of P. aeruginosa by macrophages, in which a toxic metal boost is discharged into the phagolysosome to intoxicate microbes. Importantly, we demonstrated that the regulatory link between induction of the CzcCBA system and the repression of the OprD porin responsible for carbapenem antibiotic resistance, is maintained in the macrophage environment.

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences.

RNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

Chemo-Diversification of Plant Extracts Using a Generic Bromination Reaction and Monitoring by Metabolite Profiling.

A generic procedure for direct bromination of polyphenol in crude plant extracts was developed to generate multiple "unnatural" halogenated natural products for further bioassay evaluation. To better control the halogenation procedure, the bromination was optimized with a flavonoid standard, and the reactions were monitored by high-performance liquid chromatography photometric diode array coupled to the evaporative light scattering detection (ELSD). ELSD detection was successfully used for a relative yield estimation of the compounds obtained. From the halogenation of hesperitin (11), five brominated compounds were obtained. After optimization, the reaction was successfully applied to the methanolic extract of Citrus sinensis peels, a typical waste biomass and also to the methanolic extract of the medicinal plant Curcuma longa. In both cases, the methanolic extracts were profiled by NMR for a rapid estimation of the polyphenol versus primary metabolite content. An enriched secondary metabolites extract was obtained using vacuum liquid chromatography and submitted to bromination. Metabolite profiling performed by ultrahigh purity liquid chromatography time-of-flight high-resolution mass spectrometry revealed the presence of various halogenated products. To isolate these compounds, the reactions were scaled up, and six halogenated analogues were isolated and fully characterized by NMR and high-resolution electrospray ionization mass spectrometry analyses. The antibacterial properties of these compounds were evaluated using in vitro bioassays against multiresistant strains of Staphylococcus aureus and Pseudomonas aeruginosa. Some of the halogenated derivatives obtained presented moderate antibacterial properties.

Pseudomonas aeruginosa zinc homeostasis: Key issues for an opportunistic pathogen.

Zinc is an essential trace element for almost all living organisms. In the opportunistic bacterial pathogen Pseudomonas aeruginosa, zinc has been shown to play an important role in virulence, in colonization of the host organism and has also been shown to be involved in antibiotic resistance. P. aeruginosa possesses numerous systems enabling it to thrive in zinc-depleted conditions as well as high-zinc situations, two environments that are encountered during human infection. These capabilities account for its pathogenic strength. The main aim of this review is to focus on zinc homeostasis in P. aeruginosa and the genetic regulation of the systems involved. The interconnection with virulence, as well as the mechanism of co-regulation between metal and antibiotic resistance, are of prime interest for understanding the molecular mechanisms allowing P. aeruginosa to switch from its existence as a common environmental bacterium to a severe opportunistic pathogen. This article is part of a Special Issue entitled: Dynamic gene expression, edited by Prof. Patrick Viollier.

Transcriptome Analysis of Pseudomonas aeruginosa Cultured in Human Burn Wound Exudates.

Pseudomonas aeruginosa is a severe opportunistic pathogen and is one of the major causes of hard to treat burn wound infections. Herein we have used an RNA-seq transcriptomic approach to study the behavior of P. aeruginosa PAO1 growing directly on human burn wound exudate. A chemical analysis of compounds used by this bacterium, coupled with kinetics expression of central genes has allowed us to obtain a global view of P. aeruginosa physiological and metabolic changes occurring while growing on human burn wound exudate. In addition to the numerous virulence factors and their secretion systems, we have found that all iron acquisition mechanisms were overexpressed. Deletion and complementation with pyoverdine demonstrated that iron availability was a major limiting factor in burn wound exudate. The quorum sensing systems, known to be important for the virulence of P. aeruginosa, although moderately induced, were activated even at low cell density. Analysis of bacterial metabolism emphasized importance of lactate, lipid and collagen degradation pathways. Overall, this work allowed to designate, for the first time, a global view of P. aeruginosa characteristics while growing in human burn wound exudate and highlight the possible therapeutic approaches to combat P. aeruginosa burn wound infections.