Synthesis and Evaluation of a Self-Adjuvanting Pseudomonal Vaccine Based on Major Outer Membrane Porin OprF Epitopes Formulated with Low-Toxicity QS-21-Containing Liposomes.

Pseudomonas aeruginosa (PA) is a Gram-negative pathogen that the World Health Organization has ranked as a priority 1 (critical) threat. One potential prophylactic approach to preventing or reducing the incidence of PA would be development of a long sought-after vaccine. Both antibody and CD4+ T-cell responses have been noted as playing key roles in protection against infection. In these studies, we have designed a prototype vaccine consisting of several known linear B-cell epitopes derived from an outer membrane porin F (OprF). The resulting thiol-containing protein was conjugated to a version of the lipopeptide-based Toll-like receptor agonist Pam3CysSK4Mal (10) containing a maleimide moiety and formulated into dipalmitoylphosphatidylcholine (DPPC)/cholesterol (Chol) liposomes. Mice immunized with the resulting vaccine generated antibodies that bound PA14 (serotype O10) in vitro and induced opsonization in the presence of rabbit complement and murine macrophage RAW264.7 cells. The liposome was optimized to contain 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), Chol, Pam3CysSK4-OprF (12) and the Quillaja saponaria-derived saponin adjuvant QS-21. The resulting vaccine formulation produced significantly higher antibody titers, increased the IgG2a antibody isotype, and increased the number of IgG-producing B-cells as well as splenic primed T-cells. In summary, the liposomal vaccine platform was found highly useful for the generation of a robust and balanced TH1/TH2 response.

Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa.

The tRNA (m1G37) methyltransferase TrmD catalyzes m1G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa (PaTrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of PaTrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as PaTrmD substrates. Analysis of steady-state kinetics with S-adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that PaTrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to PaTrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM (Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA (Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric PaTrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into PaTrmD as a potential target for the development of antibiotics.

Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa.

Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNAMet(CAU) and tRNATrp(CCA) are substrates for Cm formation, tRNAGln(UUG), tRNAPro(UGG), tRNAPro(CGG) and tRNAHis(GUG) for Um, and tRNAPro(GGG) for Am. tRNASer(UGA), previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNAPro(GGG) and Um in tRNAGln(UUG) by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response.

Chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease.

Inflammatory bowel disease (IBD) arises from inappropriate activation of the mucosal immune system resulting in a state of chronic inflammation with causal links to colon cancer. Helicobacter hepaticus-infected Rag2(-/-) mice emulate many aspects of human IBD, and our recent work using this experimental model highlights the importance of neutrophils in the pathology of colitis. To define molecular mechanisms linking colitis to the identity of disease biomarkers, we performed a translational comparison of protein expression and protein damage products in tissues of mice and human IBD patients. Analysis in inflamed mouse colons identified the neutrophil- and macrophage-derived damage products 3-chlorotyrosine (Cl-Tyr) and 3-nitrotyrosine, both of which increased with disease duration. Analysis also revealed higher Cl-Tyr levels in colon relative to serum in patients with ulcerative colitis and Crohn disease. The DNA chlorination damage product, 5-chloro-2'-deoxycytidine, was quantified in diseased human colon samples and found to be present at levels similar to those in inflamed mouse colons. Multivariate analysis of these markers, together with serum proteins and cytokines, revealed a general signature of activated innate immunity in human IBD. Signatures in ulcerative colitis sera were strongly suggestive of neutrophil activity, and those in Crohn disease and mouse sera were suggestive of both macrophage and neutrophil activity. These data point to innate immunity as a major determinant of serum and tissue profiles and provide insight into IBD disease processes.

DNA methylation impacts gene expression and ensures hypoxic survival of Mycobacterium tuberculosis.

DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N6-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces the expression of a number of genes. Each has a MamA site located at a conserved position relative to the sigma factor -10 binding site and transcriptional start site, suggesting that MamA modulates their expression through a shared, not locus-specific, mechanism. While strains lacking MamA grow normally in vitro, they are attenuated in hypoxic conditions, suggesting that methylation promotes survival in discrete host microenvironments. Interestingly, we demonstrate strikingly different patterns of DNA methyltransferase activity in different lineages of M. tuberculosis, which have been associated with preferences for distinct host environments and different disease courses in humans. Thus, MamA is the major functional adenine methyltransferase in M. tuberculosis strains of the Euro-American lineage while strains of the Beijing lineage harbor a point mutation that largely inactivates MamA but possess a second functional DNA methyltransferase. Our results indicate that MamA influences gene expression in M. tuberculosis and plays an important but strain-specific role in fitness during hypoxia.

A multidimensional platform for the purification of non-coding RNA species.

A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes.

MALDI-TOF imaging analysis of benzalkonium chloride penetration in ex vivo human skin.

Benzalkonium chloride (BZK), alkyldimethylbenzlamonium chloride, is a cationic surfactant that is used as an antiseptic. BZK is classified as a quaternary ammonium compound composed of molecules of several alkyl chains of differing lengths, that dictate its effectiveness towards different microbes. As a result, BZK has become one of the most used preservatives in antibacterial solutions. Despite its widespread use, it is not clear whether BZK penetrates human skin. To answer this question, BZK treated skin was analyzed using matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry imaging. Solutions containing BZK and differing excipients, including citric acid, caprylyl glycol, and vitamin E, were applied ex vivo to excised human skin using Franz diffusion cells. Treated skin was embedded in gelatin and sectioned prior to MALDI-TOF imaging. BZK penetrates through the epidermis and into the dermis, and the penetration depth was significantly altered by pH and additives in tested solutions.

Increased levels of inosine in a mouse model of inflammation.

One possible mechanism linking inflammation with cancer involves the generation of reactive oxygen, nitrogen, and halogen species by activated macrophages and neutrophils infiltrating sites of infection or tissue damage, with these chemical mediators causing damage that ultimately leads to cell death and mutation. To determine the most biologically deleterious chemistries of inflammation, we previously assessed products across the spectrum of DNA damage arising in inflamed tissues in the SJL mouse model nitric oxide overproduction ( Pang et al. ( 2007 ) Carcinogenesis 28 , 1807 - 1813 ). Among the anticipated DNA damage chemistries, we observed significant changes only in lipid peroxidation-derived etheno adducts. We have now developed an isotope-dilution, liquid chromatography-coupled, tandem quadrupole mass spectrometric method to quantify representative species across the spectrum of RNA damage products predicted to arise at sites of inflammation, including nucleobase deamination (xanthosine and inosine), oxidation (8-oxoguanosine), and alkylation (1,N(6)-ethenoadenosine). Application of the method to the liver, spleen, and kidney from the SJL mouse model revealed generally higher levels of oxidative background RNA damage than was observed in DNA in control mice. However, compared to control mice, RcsX treatment to induce nitric oxide overproduction resulted in significant increases only in inosine and only in the spleen. Further, the nitric oxide synthase inhibitor, N-methylarginine, did not significantly affect the levels of inosine in control and RcsX-treated mice. The differences between DNA and RNA damage in the same animal model of inflammation point to possible influences from DNA repair, RcsX-induced alterations in adenosine deaminase activity, and differential accessibility of DNA and RNA to reactive oxygen and nitrogen species as determinants of nucleic acid damage during inflammation.

Comparative analysis of four oxidized guanine lesions from reactions of DNA with peroxynitrite, singlet oxygen, and γ-radiation.

Oxidative damage to DNA has many origins, including irradiation, inflammation, and oxidative stress, but the chemistries are not the same. The most oxidizable base in DNA is 2-deoxyguanosine (dG), and the primary oxidation products are 8-oxodG and 2-amino-imidazolone. The latter rapidly converts to 2,2-diamino-oxazolone (Ox), and 8-oxodG is further oxidized to spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). In this study, we have examined the dose-response relationship for the formation of the above four products arising in calf thymus DNA exposed to gamma irradiation, photoactivated rose bengal, and two sources of peroxynitrite. In order to carry out these experiments, we developed a chromatographic system and synthesized isotopomeric internal standards to enable accurate and precise analysis based upon selected reaction monitoring mass spectrometry. 8-OxodG was the most abundant products in all cases, but its accumulation was highly dependent on the nature of the oxidizing agent and the subsequent conversion to Sp and Gh. Among the other oxidation products, Ox was the most abundant, and Sp was formed in significantly greater yield than Gh.

Ribonucleosides from tRNA in hyperglycemic mammalian cells and diabetic murine cardiac models.

AIMS: Cardiomyopathy is a diabetic comorbidity with few molecular targets. To address this, we evaluated transfer RNA (tRNA) modifications in the diabetic heart because tRNA modifications have been implicated in diabetic etiologies. MAIN METHODS: tRNA was isolated from aorta, apex, and atrial tissue of healthy and diabetic murine hearts and related hyperglycemic cell models. tRNA modifications and canonical ribonucleosides were quantified by liquid-chromatography tandem mass spectrometry (LC-MS/MS) using stable isotope dilution. Correlations between ribonucleosides and diabetic comorbidity pathology were assessed using statistical analyses. KEY FINDINGS: Total tRNA ribonucleoside levels were analyzed from cell types and healthy and diabetic murine heart tissue. Each heart structure had characteristic ribonucleoside profiles and quantities. Several ribonucleosides were observed as significantly different in hyperglycemic cells and diabetic tissues. In hyperglycemic models, ribonucleosides N4-acetylcytidine (ac4C), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methylcytidine (m5C), and N1-methylguanosine (m1G) were anomalous. Specific tRNA modifications known to be on murine tRNAIni(CAU) were higher in diabetic heart tissue which suggests that tRNA modifications could be regulating translation in diabetes. SIGNIFICANCE: We identified tRNA ribonucleosides and tRNA species associated with hyperglycemia and diabetic etiology.

Comparison of Small Biomolecule Ionization and Fragmentation in Pseudomonas aeruginosa Using Common MALDI Matrices.

Different bacterial cell surface associated biomolecules can be analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and coupled with collision induced dissociation (CID) for identification. Pseudomonas aeruginosa is an opportunistic, Gram-negative bacterium that causes acute or chronic biofilm infections. Cells of P. aeruginosa communicate through a system of signaling biomolecules known as quorum sensing (QS). The QS system can result in the production of biosurfactant rhamnolipids known to associate and alter the cellular membrane. MALDI-TOF utilizes a variety of matrices that can interact differently with biomolecules for selective ionization. We examined six common matrices to determine the optimal matrix specific to different molecule classes in P. aeruginosa associated with cell surfaces. Three major molecule classes (quinolones, rhamnolipids, and phospholipids) were observed to ionize selectively with the different matrices tested. Sodiated and protonated adducts differed between matrices utilized in our study. Isobaric ions were identified as different molecule classes depending on the matrix used. We highlight the role of matrix selection in MALDI-TOF identification of molecules within a complex biological mixture.

Characterization of Distinct Biofilm Cell Subpopulations and Implications in Quorum Sensing and Antibiotic Resistance.

Bacteria change phenotypically in response to their environment. Free swimming cells transition to biofilm communities that promote cellular cooperativity and resistance to stressors and antibiotics. We uncovered three subpopulations of cells with diverse phenotypes from a single-species Pseudomonas aeruginosa PA14 biofilm, and used a series of steps to isolate, characterize, and map these cell subpopulations in a biofilm. The subpopulations were distinguishable by size and morphology using dynamic light scattering (DLS) and scanning electron microscopy (SEM). Additionally, growth and dispersal of biofilms originating from each cell subpopulation exhibited contrasting responses to antibiotic challenge. Cell subpopulation surface charges were distinctly different, which led us to examine the ionizable surface molecules associated with each subpopulation using mass spectrometry. Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of cell subpopulations revealed ions unique to each subpopulation of cells that significantly co-localized with ions associated with quorum sensing. Transcript levels of algR, lasR, and rhlI in subpopulations isolated from biofilms differed from levels in planktonic stationary and mid-log cell subpopulations. These studies provide insight into diverse phenotypes, morphologies, and biochemistries of PA14 cell subpopulations for potential applications in combating bacterial pathogenesis, with medical, industrial, and environmental complications. IMPORTANCE Pseudomonas aeruginosa biofilms can cause chronic infections in burn wounds, grow on medical equipment, and proliferate in the lungs of people with cystic fibrosis. These inherently antibiotic tolerant biofilms are difficult to eradicate largely due to the complexity of the biofilm environment. Developing more effective biofilm treatments is reliant upon understanding biofilm heterogeneity. We identified and characterized three separate cell subpopulations found in P. aeruginosa PA14 biofilms. The distinct morphologies, phenotypes, and biochemistries of each of these cell subpopulations indicate that they contribute differently to the overall biofilm environment. These findings demonstrate that bacterial cells of the same species exhibit diversity that implies distinct roles in biofilm initiation, maturation, and maintenance.

Oxidative DNA strand scission induced by peptides.

Cellular oxidative stress promotes chemical reactions causing damage to DNA, proteins, and membranes. Here, we describe experiments indicating that reactive oxygen species, in addition to degrading polypeptides and polynucleotides through direct reactions, can also promote damaging biomolecular cross reactivity by converting protein residues into peroxides that cleave the DNA backbone. The studies reported show that a variety of residues induce strand scission upon oxidation, and hydrogen abstraction occurring at the DNA backbone is responsible for the damage. The observation of peptide-promoted DNA damage suggests that crossreactions within protein/DNA complexes should be considered as a significant cause of the toxicity of reactive oxygen species.

A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA: Application to Stress-Induced Reprogramming of tRNA Modifications.

Here we describe an analytical platform for systems-level quantitative analysis of modified ribonucleosides in any RNA species, with a focus on stress-induced reprogramming of tRNA as part of a system of translational control of cell stress response. This chapter emphasizes strategies and caveats for each of the seven steps of the platform workflow: (1) RNA isolation, (2) RNA purification, (3) RNA hydrolysis to individual ribonucleosides, (4) chromatographic resolution of ribonucleosides, (5) identification of the full set of modified ribonucleosides, (6) mass spectrometric quantification of ribonucleosides, (6) interrogation of ribonucleoside datasets, and (7) mapping the location of stress-sensitive modifications in individual tRNA molecules. We have focused on the critical determinants of analytical sensitivity, specificity, precision, and accuracy in an effort to ensure the most biologically meaningful data on mechanisms of translational control of cell stress response. The methods described here should find wide use in virtually any analysis involving RNA modifications.

Photosensitized DNA cleavage promoted by amino acids.

A novel class of DNA cleavage agents are reported that derive activity from amino acids tethered to a photoactive intercalator.

Challenges in developing DNA and RNA biomarkers of inflammation.

Inflammation is now a proven cause of human diseases such as cancer and cardiovascular disease. One potential link between inflammation and disease involves secretion of reactive chemical species by immune cells, with chronic damage to host epithelial cells leading to disease. This suggests pathophysiologically that DNA and RNA damage products are candidate biomarkers of inflammation, both for mechanistic understanding of the process and for risk assessment. Of the current approaches to quantifying DNA damage products, mass spectrometry-based methods provide the most rigorous quantification needed for biomarker development, while antibody-based approaches provide the most practical way to implement biomarkers in a clinical setting. Nonetheless, all approaches are biased by adventitious formation of DNA and RNA damage products during sample processing. Recent studies of tissue-derived DNA biomarkers in mouse models of inflammation reveal significant changes only in DNA adducts derived from lipid peroxidation. These and other observations raise the question of the most appropriate sampling compartment for DNA biomarker studies and highlight the emerging role of lipid damage in inflammation.

Tunable DNA cleavage by intercalating peptidoconjugates.

The properties of a novel family of peptide-based DNA-cleavage agents are described. Examination of the DNA-cleavage activities of a systematic series of peptide-intercalator conjugates revealed trends that show a strong dependence on peptide sequence. Conjugates differing by a single residue displayed reactivities that varied over a wide range. The cleavage activity was modulated by the electrostatic or steric qualities of individual amino acids. Isomeric conjugates that differed in the position of the tether also exhibited different reactivities. The mechanism of DNA cleavage for these compounds was also probed and was determined to involve hydrogen-atom abstraction from the DNA backbone. Previous studies of these compounds indicated that amino acid peroxides were the active agents in the cleavage reaction; in this report, the chemistry underlying the reaction is characterized. The results reported provide insight into how peptide sequences can be manipulated to produce biomimetic compounds.