Search

Hydrogen sulfide (H 2 S) has been proposed to protect bacteria from antibiotics, pointing to H 2 S-producing enzymes as possible targets for the development of antibiotic adjuvants. Here, MIC assays performed with Pseudomonas aeruginosa mutants producing altered H 2 S levels demonstrate that H 2 S does not affect antibiotic resistance in this bacterium. Moreover, correlation analyses in a large collection of P. aeruginosa cystic fibrosis isolates argue against the protective role of H 2 S from antibiotic activity during chronic lung infection., Competing Interests: The authors declare no conflict of interest.

Hydrogen sulfide (H 2 S) and nitric oxide (NO) are long-known inhibitors of terminal oxidases in the respiratory chain. Yet, they exert pivotal signaling roles in physiological processes, and in several bacterial pathogens have been reported to confer resistance against oxidative stress, host immune responses, and antibiotics. Pseudomonas aeruginosa , an opportunistic pathogen causing life-threatening infections that are difficult to eradicate, has a highly branched respiratory chain including four terminal oxidases of the haem-copper type ( aa 3 , cbb 3 -1 , cbb 3 -2 , and bo 3 ) and one oxidase of the bd -type (cyanide-insensitive oxidase, CIO). As Escherichia coli bd -type oxidases have been shown to be H 2 S-insensitive and to readily recover their activity from NO inhibition, here we tested the effect of H 2 S and NO on CIO by performing oxygraphic measurements on membrane preparations from P. aeruginosa PAO1 and isogenic mutants depleted of CIO only or all other terminal oxidases except CIO. We show that O 2 consumption by CIO is unaltered even in the presence of high levels of H 2 S, and that CIO expression is enhanced and supports bacterial growth under such stressful conditions. In addition, we report that CIO is reversibly inhibited by NO, while activity recovery after NO exhaustion is full and fast, suggesting a protective role of CIO under NO stress conditions. As P. aeruginosa is exposed to H 2 S and NO during infection, the tolerance of CIO towards these stressors agrees with the proposed role of CIO in P. aeruginosa virulence.

In Pseudomonas aeruginosa relevant features including virulence and biofilm formation are controlled by quorum sensing (QS), a cell density-dependent intercellular communication system based on the production and response to signal molecules. P. aeruginosa has evolved chemically distinct compounds employed as QS signal molecules (QSSMs) that can be detected and quantified through rapid, sensitive, and low-cost methods based on whole-cell biosensors. Here, we present a series of protocols based on whole-cell biosensors for qualitative and quantitative analysis of QSSMs produced by P. aeruginosa. These protocols can be used to investigate the impact of environmental conditions, genetic modifications, or quorum quenching agents on the production of QSSMs in P. aeruginosa., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Single-cell analyses can reveal that despite experiencing identical physico-chemical conditions, individual bacterial cells within a monoclonal population may exhibit variations in gene expression. Such phenotypic heterogeneity has been described for several aspects of bacterial physiology, including QS activation. This study demonstrates that the transition of non-quorate cells to the quorate state is a graded process that does not occur at a specific cell density and that subpopulations of non-quorate cells also persist at high cell density. Here, we provide a mechanistic explanation for this phenomenon, showing that a negative feedback regulatory loop integrated into the las system has a pivotal role in promoting cell-to-cell variation in the QS activation state and in limiting the transition of non-quorate cells to the quorate state in P. aeruginosa ., Competing Interests: The authors declare no conflict of interest.

Introduction: The increase in bacterial strains resistant to conventional antibiotics is an alarming problem for human health and could lead to pandemics in the future. Among bacterial pathogens responsible for a large variety of severe infections there is Pseudomonas aeruginosa . Therefore, there is an urgent need for new molecules with antimicrobial activity or that can act as adjuvants of antibiotics already in use. In this scenario, antimicrobial peptides (AMPs) hold great promise. Recently, we characterized a frog-skin AMP derived from esculentin-1a, namely Esc(1-21)-1c, endowed with antipseudomonal activity without being cytotoxic to human cells. Methods: The combinatorial effect of the peptide and antibiotics was investigated through the checkerboard assay, differential proteomic and transcriptional analysis. Results: Here, we found that Esc(1-21)-1c can synergistically inhibit the growth of P. aeruginosa cells with three different antibiotics, including tetracycline. We therefore investigated the underlying mechanism implemented by the peptide using a differential proteomic approach. The data revealed a significant decrease in the production of three proteins belonging to the MexAB-OprM efflux pump upon treatment with sub-inhibitory concentration of Esc(1-21)-1c. Down-regulation of these proteins was confirmed by transcriptional analysis and direct measurement of their relative levels in bacterial cells by tandem mass spectrometry analysis in multiple reaction monitoring scan mode. Conclusion: These evidences suggest that treatment with Esc(1-21)-1c in combination with antibiotics would increase the intracellular drug content making bacteria more susceptible to the antibiotic. Overall, these results highlight the importance of characterizing new molecules able to synergize with conventional antibiotics, paving the way for the development of alternative therapeutic strategies based on AMP/antibiotic formulations to counteract the emergence of resistant bacterial strains and increase the use of "old" antibiotics in medical practice., Competing Interests: Author ADS was employed by CEINGE Biotecnologie Avanzate. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Canè, Casciaro, Di Somma, Loffredo, Puglisi, Battaglia, Mellini, Cappiello, Rampioni, Leoni, Amoresano, Duilio and Mangoni.)

Pseudomonas aeruginosa is a model quorum sensing (QS) pathogen with three interconnected QS circuits that control the production of virulence factors and antibiotic tolerant biofilms. The pqs QS system of P. aeruginosa is responsible for the biosynthesis of diverse 2-alkyl-4-quinolones (AQs), of which 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4( 1H )-quinolone (PQS) function as QS signal molecules. Transcriptomic analyses revealed that HHQ and PQS influenced the expression of multiple genes via PqsR-dependent and -independent pathways whereas 2-heptyl-4-hydroxyquinoline N -oxide (HQNO) had no effect on P. aeruginosa transcriptome. HQNO is a cytochrome bc 1 inhibitor that causes P. aeruginosa programmed cell death and autolysis. However, P. aeruginosa pqsL mutants unable to synthesize HQNO undergo autolysis when grown as colony biofilms. The mechanism by which such autolysis occurs is not understood. Through the generation and phenotypic characterization of multiple P. aeruginosa PAO1 mutants producing altered levels of AQs in different combinations, we demonstrate that mutation of pqsL results in the accumulation of HHQ which in turn leads to Pf4 prophage activation and consequently autolysis. Notably, the effect of HHQ on Pf4 activation is not mediated via its cognate receptor PqsR. These data indicate that the synthesis of HQNO in PAO1 limits HHQ-induced autolysis mediated by Pf4 in colony biofilms. A similar phenomenon is shown to occur in P. aeruginosa cystic fibrosis (CF) isolates, in which the autolytic phenotype can be abrogated by ectopic expression of pqsL ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Giallonardi, Letizia, Mellini, Frangipani, Halliday, Heeb, Cámara, Visca, Imperi, Leoni, Williams and Rampioni.)

In the opportunistic pathogen Pseudomonas aeruginosa, many virulence traits are finely regulated by quorum sensing (QS), an intercellular communication system that allows the cells of a population to coordinate gene expression in response to cell density. The key aspects underlying the functionality of the complex regulatory network governing QS in P. aeruginosa are still poorly understood, including the interplay between the effector protein PqsE and the transcriptional regulator RhlR in controlling the QS regulon. Different studies have focused on the characterization of PqsE- and RhlR-controlled genes in genetic backgrounds in which RhlR activity can be modulated by PqsE and pqsE expression is controlled by RhlR, thus hampering identification of the distinct regulons controlled by PqsE and RhlR. In this study, a P. aeruginosa PAO1 mutant strain with deletion of multiple QS elements and inducible expression of pqsE and/or rhlR was generated and validated. Transcriptomic analyses performed on this genetic background allowed us to unambiguously define the regulons controlled by PqsE and RhlR when produced alone or in combination. Transcriptomic data were validated via reverse transcription-quantitative PCR (RT-qPCR) and transcriptional fusions. Overall, our results showed that PqsE has a negligible effect on the P. aeruginosa transcriptome in the absence of RhlR, and that multiple RhlR subregulons exist with distinct dependency on PqsE. Overall, this study contributes to untangling the regulatory link between the pqs and rhl QS systems mediated by PqsE and RhlR and clarifying the impact of these QS elements on the P. aeruginosa transcriptome. IMPORTANCE The ability of Pseudomonas aeruginosa to cause difficult-to-treat infections relies on its capacity to fine-tune the expression of multiple virulence traits via the las , rhl , and pqs QS systems. Both the pqs effector protein PqsE and the rhl transcriptional regulator RhlR are required for full production of key virulence factors in vitro and pathogenicity in vivo . While it is known that PqsE can stimulate the ability of RhlR to control some virulence factors, no data are available to allow clear discrimination of the PqsE and RhlR regulons. The data produced in this study demonstrate that PqsE mainly impacts the P. aeruginosa transcriptome via an RhlR-dependent pathway and splits the RhlR regulon into PqsE-dependent and PqsE-independent subregulons. Besides contributing to untangling of the complex QS network of P. aeruginosa, our data confirm that both PqsE and RhlR are suitable targets for the development of antivirulence drugs.

Key microbial processes in many bacterial species are heterogeneously expressed in single cells of bacterial populations. However, the paucity of adequate molecular tools for live, real-time monitoring of multiple-gene expression at the single-cell level has limited the understanding of phenotypic heterogeneity. To investigate phenotypic heterogeneity in the ubiquitous opportunistic pathogen Pseudomonas aeruginosa , a genetic tool that allows gauging multiple-gene expression at the single-cell level has been generated. This tool, named pRGC, consists of a promoter-probe vector for transcriptional fusions that carries three reporter genes coding for the fluorescent proteins mCherry, green fluorescent protein (GFP), and cyan fluorescent protein (CFP). The pRGC vector has been characterized and validated via single-cell gene expression analysis of both constitutive and iron-regulated promoters, showing clear discrimination of the three fluorescence signals in single cells of a P. aeruginosa population without the need for image processing for spectral cross talk correction. In addition, two pRGC variants have been generated for either (i) integration of the reporter gene cassette into a single neutral site of P. aeruginosa chromosome that is suitable for long-term experiments in the absence of antibiotic selection or (ii) replication in bacterial genera other than Pseudomonas The easy-to-use genetic tools generated in this study will allow rapid and cost-effective investigation of multiple-gene expression in populations of environmental and pathogenic bacteria, hopefully advancing the understanding of microbial phenotypic heterogeneity. IMPORTANCE Within a bacterial population, single cells can differently express some genes, even though they are genetically identical and experience the same chemical and physical stimuli. This phenomenon, known as phenotypic heterogeneity, is mainly driven by gene expression noise and results in the emergence of bacterial subpopulations with distinct phenotypes. The analysis of gene expression at the single-cell level has shown that phenotypic heterogeneity is associated with key bacterial processes, including competence, sporulation, and persistence. In this study, new genetic tools have been generated that allow easy cloning of up to three promoters upstream of distinct fluorescent genes, making it possible to gauge multiple-gene expression at the single-cell level by fluorescence microscopy without the need for advanced image-processing procedures. A proof of concept has been provided by investigating iron uptake and iron storage gene expression in response to iron availability in P. aeruginosa ., (Copyright © 2021 American Society for Microbiology.)

Enzymes are essential to maintain organisms alive. Some of the reactions they catalyze are associated with a change in reagents chirality, hence their activity can be tracked by using optical means. However, illumination affects enzyme activity: the challenge is to operate at low-intensity regime avoiding loss in sensitivity. Here we apply quantum phase estimation to real-time measurement of invertase enzymatic activity. Control of the probe at the quantum level demonstrates the potential for reducing invasiveness with optimized sensitivity at once. This preliminary effort, bringing together methods of quantum physics and biology, constitutes an important step towards full development of quantum sensors for biological systems.

The emergence of antibiotic resistant bacterial pathogens is increasing at an unprecedented pace, calling for the development of new therapeutic options. Small molecules interfering with virulence processes rather than growth hold promise as an alternative to conventional antibiotics. Anti-virulence agents are expected to decrease bacterial virulence and to pose reduced selective pressure for the emergence of resistance. In the opportunistic pathogen Pseudomonas aeruginosa the expression of key virulence traits is controlled by quorum sensing (QS), an intercellular communication process that coordinates gene expression at the population level. Hence, QS inhibitors represent promising anti-virulence agents against P. aeruginosa . Virtual screenings allow fast and cost-effective selection of target ligands among vast libraries of molecules, thus accelerating the time and limiting the cost of conventional drug-discovery processes, while the drug-repurposing approach is based on the identification of off-target activity of FDA-approved drugs, likely endowed with low cytotoxicity and favorable pharmacological properties. This study aims at combining the advantages of virtual screening and drug-repurposing approaches to identify new QS inhibitors targeting the pqs QS system of P. aeruginosa . An in silico library of 1,467 FDA-approved drugs has been screened by molecular docking, and 5 hits showing the highest predicted binding affinity for the pqs QS receptor PqsR (also known as MvfR) have been selected. In vitro experiments have been performed by engineering ad hoc biosensor strains, which were used to verify the ability of hit compounds to decrease PqsR activity in P. aeruginosa . Phenotypic analyses confirmed the impact of the most promising hit, the antipsychotic drug pimozide, on the expression of P. aeruginosa PqsR-controlled virulence traits. Overall, this study highlights the potential of virtual screening campaigns of FDA-approved drugs to rapidly select new inhibitors of important bacterial functions., (Copyright © 2019 Mellini, Di Muzio, D’Angelo, Baldelli, Ferrillo, Visca, Leoni, Polticelli and Rampioni.)

Calcium sulphate dihydrate nanocrystals of 25-100 nm width have been synthesized in 100% purity and yield by means of a method--the cryo-vacuum process--consisting of rapid freezing of quasi-saturated solutions and subsequent vacuum assisted sublimation of water. Transmission electron microscopy reveals both curled nano-lamellae and smaller, irregular particles; electron diffraction patterns demonstrated that the particles are crystalline. This is a very powerful method for the 'clean' synthesis of moderately and completely water-soluble inorganic materials.

A practical, offline method for experimental detection and correction for projector lens distortion in the transmission electron microscope (TEM) operating in high-resolution (HR) and selected area electron diffraction (SAED) modes is described. Typical TEM works show that, in the simplest case, the distortion transforms on the recording device, which would be a circle into an ellipse. The first goal of the procedure described here is to determine the elongation and orientation of the ellipse. The second goal is to correct for the distortion using an ordinary graphic program. The same experimental data set may also be used to determine the actual microscope magnification and the rotation between SAED patterns and HR images. The procedure may be helpful in several quantitative applications of electron diffraction and HR imaging, for instance while performing accurate lattice parameter determination, or while determining possible metrical deviations (cell edges and angles) from a given symmetry.

A depleted-uranium penetrator, shot in 1999 at Djakovica, Western Kosovo, and there collected in June 2001, shows evident alteration processes, perceivable as black and yellow coatings. XRD indicates that the black coating mostly consists of uraninite, UO2, with possible presence of other more oxidized uranium forms, such as U3O8. The yellow material is mostly amorphous, with variable weak diffraction lines, due to minor embedded uraninite grains, or possibly to schoepite, UO3 x 2H2O. SEM-EDS reveals only uranium. Whereas uraninite does not show any crystal shape, the yellow material recrystallizes to flattened pseudo-hexagonal prisms, approximately 2-10 microm wide and 1-4 microm long. Raman spectra of the yellow material have peaks at 3474 and 3222 cm(-1), indicative for OH groups, plus at 812 and 744 cm(-1), indicative for UO2(2+) uranyl ions. Based on the different data, the yellow material covering the depleted-uranium dart is an oxidized corrosion product, containing uranyl ions and hydroxyls and/or water molecules, akin to schoepite. Therefore, the Djakovica dart shows evident oxidation and leaching processes, progressively releasing mobile uranium forms. As uranium will be progressively dispersed far from the impact sites, at a rate controlled by the presence of effective fixing mechanisms, we feel necessary to maintain long term geochemical control of water pollution within the battlefield surroundings.

Authors reports the case of a seven months toddler with transient hyperphosphatasemia without clinical manifestations and no other hepatic or bone disfunctions. Resolution of both enzymes and bone isoenzymes occurs within 5 months.

Neurofibrillary tangles (NFT), one of the neurodegenerative features of Alzheimer's disease, Down's syndrome and normal aging, is a constant, widespread neuropathological finding in Guamanian amyotrophic lateral sclerosis (ALS), parkinsonism-dementia (PD) and in neurologically normal Guamanians, dying of causes other than ALS and PD. NFT in brain tissue sections of patients with Guamanian ALS and PD were immunoreactive to antibodies directed against a 43-amino acid synthetic peptide homologous to amyloid beta/A4-protein (anti-SP43) associated with Alzheimer's disease. NFT extracted from frozen brain tissues of Guamanian patients with ALS and PD and from tissues of neurologically normal Guamanians were congophilic and birefringent. By negative-stain electron microscopy, NFT preparations contained bundles and/or isolated single, straight, unpaired filaments in Guamanian ALS and occasionally pairing of filaments in neurologically normal Guamanians, measuring 5-20 nm in diameter. Formic acid digestion of NFT preparations, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion high-pressure liquid chromatography, showed a protein with an apparent molecular mass of 4- to 4.5-kDa, which by Western blot analysis was immunoreactive to anti-SP43. Immunoabsorption of purified NFT or SP43 with anti-SP43 abolished immunostaining. Our study corroborate previous data that amyloid beta/A4-protein is present in NFT in Guamanian PD. Furthermore, our data indicate that amyloid beta/A4-protein is present in NFT in brain tissues of patients with Guamanian ALS and in neurologically normal Guamanians, suggesting a common mechanism of amyloidogenesis with NFT formation in Alzheimer's disease and normal brain aging.

When fully grown Xenopus oocytes are stimulated by progesterone, a period of protein synthesis is necessary for maturation. Synthesis of the mos proto-oncogene product, pp39mos, is necessary for the activation of M-phase promoting factor (MPF) in meiosis I. On the basis that mos is translated de novo on hormonal stimulation of Xenopus oocytes and that injecting mos RNA into oocytes induces their maturation, we have proposed that the mos protein is a candidate initiator of oocyte maturation, needed to trigger the conversion of precursor MPF into its active form. To determine whether mos is the only protein required for initiating maturation, we have produced a soluble, active recombinant mos protein and injected it into Xenopus oocytes. We report here that in the absence of protein synthesis that mos protein efficiently induces germinal vesicle breakdown and the activation of MPF. The oocytes, however, do not proceed into meiosis II. Thus, the mos protein fulfills the requirements of an initiator protein, but the synthesis of one or more additional proteins may be necessary to complete oocyte maturation.

The Rex protein of human T-cell leukemia virus type I (HTLV-I) was expressed in bacteria and partially purified. Rex was shown to bind in vitro specifically to an RNA sequence located in the 3' long terminal repeat of HTLV-I, named Rex-responsive element (RXRE). Rex also bound in vitro to the human immunodeficiency virus type 1 (HIV-1) Rev-responsive element (RRE), while purified HIV-1 Rev protein did not bind to the RXRE. The binding results obtained in vitro are therefore in agreement with the nonreciprocal function of Rev and Rex in vivo. Rex binds specifically to both RRE and RXRE and activates expression in both HIV-1 and HTLV-I, while Rev binds to RRE and activates only HIV-1. Binding of Rex to RRE deletion mutants previously shown to lack either the Rev-responsive or the Rex-responsive portion suggested preferential binding of Rex to a distinct target within the RRE. These results demonstrated that Rex, like Rev, acts by binding to a specific RNA target.

The binding of Rev protein of human immunodeficiency virus type 1 (HIV-1) to the cis-acting Rev-responsive element (RRE) was compared to the binding of a trans-dominant Rev mutant. RevBL, which inhibits Rev function. Rev and RevBL expressed in bacteria were purified and shown to bind in vitro to the RRE with similar affinities. The study of the RRE mutants indicated that Rev and RevBL bind to the same target within the RRE in vitro and in vivo. In vivo experiments demonstrated that RevBL did not increase the steady-state levels of HIV-1 mRNA or protein. These experiments suggested that additional cellular factors interacting with Rev but not with RevBL are necessary for function. The Rex protein of human T-cell leukemia virus type I (HTLV-I) is similar to Rev and acts through a sequence named Rex-responsive element (RXRE) located in the long terminal repeat of HTLV-I. We examined the function of RevBL on a hybrid mRNA molecule containing both the RRE and RXRE. While RevBL prevented Rev function, it did not affect Rex function on the mRNA containing either the RXRE or both the RRE and RXRE. Therefore, binding of RevBL to the RRE had neither positive nor negative effects on the mRNA, since this mRNA could be efficiently utilized in the presence of a functional Rex-RXRE interaction. The results obtained in vivo and in vitro strongly suggest that RevBL inhibits Rev function by binding to the same site as Rev and preventing Rev binding and function.

A better understanding of the structure and biochemical properties of the replicative machinery of human immunodeficiency virus type 1 (HIV-1) may be useful in the screening and design of drugs that could be used to treat AIDS. We have previously described a recombinant strain of Escherichia coli that produces HIV-1 reverse transcriptase (RT). Fermentation conditions for the large-scale growth of the bacterial strain and a protocol for the purification of an enzymatically active 66-Kd form of the RT have been developed. The purified RT has all of the appropriate enzymatic functions and properties. The recombinant protein can be substituted for the viral enzyme in structural and biochemical studies and used in screens for drugs that could inhibit HIV replication.

An evaluation of the reproducibility and accuracy of the NMR human blood test for cancer described by Fossel, E. T., Carr, J. M. and McDonagh, J., (New England Journal of Medicine 315, 1369-1376) in 1986 has been conducted jointly at the National Cancer Institute-Frederick Cancer Research Facility, Frederick, MD (NCI-FCRF) and the National Research Council, Ottawa, Canada (NRC). The influences on the test of the following were studied: (a) subject fasting; (b) sample collection, storage and handling; (c) use of plasma or serum; (d) variations of test results from the same individual with time; (e) NMR observation parameters including field strength and temperature; and (f) variations in obtaining the Fossel Index (FI) (a number defined by Fossel and co-workers as the average of the widths at half height of the regions in the NMR spectrum of human plasma at 1.3 and 0.88 ppm) by different people from the same plotted spectrum. This test was found to be reproducible but not accurate for screening a general asymptomatic population. The accuracy is defined in terms of the sensitivity, specificity, and predictive values of the test. The accuracy of the test results from our laboratories is compared with the accuracies from other laboratories including Fossel's. The correlation of the Fossel Index with total triglyceride content in the serum has been confirmed by analysing blood components using the following technologies: KBr density gradient centrifugation, high resolution agarose gel electrophoresis, high performance gel permeation chromatography, and chemical analysis.