Insights on Pseudomonas aeruginosa Carbohydrate Binding from Profiles of Cystic Fibrosis Isolates Using Multivalent Fluorescent Glycopolymers Bearing Pendant Monosaccharides.

Pseudomonas aeruginosa contributes to frequent, persistent, and, often, polymicrobial respiratory tract infections for individuals with cystic fibrosis (CF). Chronic CF infections lead to bronchiectasis and a shortened lifespan. P. aeruginosa expresses numerous adhesins, including lectins known to bind the epithelial cell and mucin glycoconjugates. Blocking carbohydrate-mediated host-pathogen and intra-biofilm interactions critical to the initiation and perpetuation of colonization offer promise as anti-infective treatment strategies. To inform anti-adhesion therapies, we profiled the monosaccharide binding of P. aeruginosa from CF and non-CF sources, and assessed whether specific bacterial phenotypic characteristics affected carbohydrate-binding patterns. Focusing at the cellular level, microscopic and spectrofluorometric tools permitted the solution-phase analysis of P. aeruginosa binding to a panel of fluorescent glycopolymers possessing distinct pendant monosaccharides. All P. aeruginosa demonstrated significant binding to glycopolymers specific for α-D-galactose, ß-D-N-acetylgalactosamine, and ß-D-galactose-3-sulfate. In each culture, a small subpopulation accounted for the binding. The carbohydrate anomeric configuration and sulfate ester presence markedly influenced binding. While this opportunistic pathogen from CF hosts presented with various colony morphologies and physiological activities, no phenotypic, physiological, or structural feature predicted enhanced or diminished monosaccharide binding. Important to anti-adhesive therapeutic strategies, these findings suggest that, regardless of phenotype or clinical source, P. aeruginosa maintain a small subpopulation that may readily associate with specific configurations of specific monosaccharides. This report provides insights into whole-cell P. aeruginosa carbohydrate-binding profiles and into the context within which successful anti-adhesive and/or anti-virulence anti-infective agents for CF must contend.

Crystal structure of (R,S)-2-hy-droxy-4-(methyl-sulfan-yl)butanoic acid.

The title compound, a major animal feed supplement, abbreviated as HMTBA and alternatively called dl-me-thio-nine hy-droxy analogue, C5H10O3S, (I), was isolated in pure anhydrous monomeric form. The melting point is 302.5 K and the compound crystallizes in the monoclinic space group P21/c, with two conformationally non-equivalent mol-ecules [(I A ) and (I B )] in the asymmetric unit. The crystal structure is formed by alternating polar and non-polar layers running along the bc plane and features an extensive hydrogen-bonding network within the polar layers. The Hirshfeld surface analysis revealed a significant contribution of non-polar H⋯H and H⋯S inter-actions to the packing forces for both mol-ecules.

Interaction of Bacterial Phenazines with Colistimethate in Bronchial Epithelial Cells.

Multidrug-resistant bacterial infections are being increasingly treated in clinics with polymyxins, a class of antibiotics associated with adverse effects on the kidney, nervous system, or airways of a significant proportion of human and animal patients. Although many of the resistant pathogens display enhanced virulence, the hazard of cytotoxic interactions between polymyxin antibiotics and bacterial virulence factors (VFs) has not been assessed, to date. We report here the testing of paired combinations of four Pseudomonas aeruginosa VF phenazine toxins, pyocyanin (PYO), 1-hydroxyphenazine (1-HP), phenazine-1-carboxylic acid (PCA), and phenazine-1-carboxamide (PCN), and two commonly prescribed polymyxin drugs, colistin-colistimethate sodium (CMS) and polymyxin B, in three human airway cell lines, BEAS-2B, HBE-1, and CFT-1. Cytotoxicities of individual antibiotics, individual toxins, and their combinations were evaluated by the simultaneous measurement of mitochondrial metabolic, total transcriptional/translational, and Nrf2 stress response regulator activities in treated cells. Two phenazines, PYO and 1-HP, were cytotoxic at clinically relevant concentrations (100 to 150 µM) and prompted a significant increase in oxidative stress-induced transcriptional activity in surviving cells. The polymyxin antibiotics arrested cell proliferation at clinically achievable (<1 mM) concentrations as well, with CMS displaying surprisingly high cytotoxicity (50% effective dose [ED50] = 180 µM) in BEAS-2B cells. The dose-response curves were probed by a median-effect analysis, which established a synergistically enhanced cytotoxicity of the PYO-CMS combination in all three airway cell lines; a particularly strong effect on BEAS-2B cells was observed, with a combination index (CI) of 0.27 at the ED50 PCA, PCN, and 1-HP potentiated CMS cytotoxicity to a smaller extent. The cytotoxicity of CMS could be reduced with 10 mM N-acetyl-cysteine. Iron chelators, while ineffective against the polymyxins, could rescue all three bronchial epithelial cell lines treated with lethal PYO or CMS-PYO doses. These findings suggest that further evaluations of CMS safety are needed, along with a search for means to moderate potentially cytotoxic interactions.

Transient Proteotoxicity of Bacterial Virulence Factor Pyocyanin in Renal Tubular Epithelial Cells Induces ER-Related Vacuolation and Can Be Efficiently Modulated by Iron Chelators.

Persistent infections of biofilm forming bacteria, such as Pseudomonas aeruginosa, are common among human populations, due to the bacterial resistance to antibiotics and other adaptation strategies, including release of cytotoxic virulent factors such as pigment pyocyanin (PCN). Urinary tract infections harbor P. aeruginosa strains characterized by the highest PCN-producing capacity, yet no information is available on PCN cytotoxicity mechanism in kidney. We report here that renal tubular epithelial cell (RTEC) line NRK-52E responds to PCN treatments with paraptosis-like activity features. Specifically, PCN-treated cells experienced dilation of endoplasmic reticulum (ER) and an extensive development of ER-derived vacuoles after about 8 h. This process was accompanied with hyper-activation of proteotoxic stress-inducible transcription factors Nrf2, ATF6, and HSF-1. The cells could be rescued by withdrawal of PCN from the culture media before the vacuoles burst and cells die of non-programmed necrosis after about 24-30 h. The paraptosis-like activity was abrogated by co-treatment of the cells with metal-chelating antioxidants. A microscopic examination of cells co-treated with PCN and agents aiming at a variety of the cellular stress mediators and pathways have identified iron as a single most significant co-factor of the PCN cytotoxicity in the RTECs. Among biologically relevant metal ions, low micromolar Fe2+ specifically mediated anaerobic oxidation of glutathione by PCN, but catechol derivatives and other strong iron complexing agents could inhibit the reaction. Our data suggest that iron chelation could be considered as a supplementary treatment in the PCN-positive infections.

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization.

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of ß-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal ß-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (≤1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired.

RAFT-based tri-component fluorescent glycopolymers: synthesis, characterization and application in lectin-mediated bacterial binding study.

A group of fluorescent statistical glycopolymers, prepared via reversible addition-fragmentation chain-transfer (RAFT)-based polymerizations, were successfully employed in lectin-mediated bacterial binding studies. The resultant glycopolymers contained three different monomers: N-(2-hydroxyethyl) acrylamide (HEAA), N-(2-aminoethyl) methacrylamide (AEMA) and N-(2-glyconamidoethyl)-methacrylamides possessing different pendant sugars. Low dispersities (≤1.32) and predictable degrees of polymerization were observed among the products. After the polymerization, the glycopolymers were further modified by different succinimidyl ester fluorophores targeting the primary amine groups on AEMA. With their binding specificities being confirmed by testing with lectin coated agarose beads, the glycopolymers were employed in bacterial binding studies, where polymers containing α-galactose or ß-galactose as the pendant sugar were specifically bound by two clinically important pathogens Pseudomonas aeruginosa and Staphylococcus aureus, respectively. This is the first report of using RAFT-based glycopolymers in bacterial binding studies, and the ready access to tri-component statistical glycopolymers also warrants further exploration of their utility in other glycobiological applications.

Quality of intrathecal baclofen from different sources.

OBJECTIVE: To compare the quality of intrathecal baclofen obtained from a national compounding pharmacy (AnazaoHealth) with the manufactured product (Lioresal) with regard to accuracy and precision of baclofen concentration, and the content of the baclofen degradation product, 4-(4-chlorophenyl)-2-pyrrolidinone (PYR). DESIGN: Samples of baclofen used for refilling intrathecal pumps were placed in 1.2-mL silicone gasket-sealed cryogenic vials and stored at or lower than -25°C. Each sample was a different lot number (Lioresal) or prescription number (AnazaoHealth). The laboratory was blinded to the source of the solutions. Coupled with electrospray ionization-mass spectrometry analyte confirmation, quantitation of baclofen and PYR in each sample was performed in duplicate by using high-performance liquid chromatography with ultraviolet detection via a photodiode array detector. SETTING: Outpatient clinic. PARTICIPANTS: Patients with intrathecal baclofen pumps. MAIN OUTCOME MEASURES: Accuracy and precision of baclofen concentration, and concentration of PYR. RESULTS: The difference of mean concentrations from expected concentrations of 500 and 2000 µg/mL were significantly greater for samples from AnazaoHealth compared with Lioresal. Values are shown as mean (± standard error), with n the number samples, for AnazaoHealth compared with Lioresal: (537.1 ± 6.7 µg/mL [n = 8] versus 515.6 ± 0.82 µg/mL [n = 5]; P = .034, respectively) and (2211.4 ± 21.6 µg/mL [n = 12] versus 2055.3 ± 8.7 µg/mL [n = 4]; P = .004, respectively). AnazaoHealth samples with expected concentration of 4000 µg/mL were 3987.7 ± 79.9 µg/mL, n = 7. All 9 Lioresal samples were within 5% of the expected concentration. Of 27 AnazaoHealth samples, 22 were more than 5%, and 8 were more than 10% different from the expected concentration. No PYR was detected in any sample from AnazaoHealth. All samples of Lioresal contained PYR, but all solutions contained less PYR than 1% of the baclofen concentration. CONCLUSIONS: Lioresal was more accurate in concentration and more precise among batches than compounded intrathecal baclofen but had higher levels of PYR.

Characterization of a unique class C acid phosphatase from Clostridium perfringens.

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Acylation of the lipooligosaccharide of Haemophilus influenzae and colonization: an htrB mutation diminishes the colonization of human airway epithelial cells.

Haemophilus influenzae is a commensal and opportunistic pathogen of the human airways. A number of surface molecules contribute to colonization of the airways by H. influenzae, such as adhesins, including structures found in the lipooligosaccharide (LOS). A human bronchiolar xenograft model was employed to investigate the host-bacterial interactions involved in the colonization of the airway by H. influenzae. Differential display was used to identify H. influenzae mRNA that reflect genes which were preferentially expressed in the xenograft compared to growth. Eleven mRNA fragments had consistent increased expression when the bacteria grew in xenografts. On sequencing these fragments, eight open reading frames were identified. Three of these had no match in the NCBI or the TIGR database, while an additional three were homologous to genes involved in heme or iron acquisition and utilization: two of the mRNAs encoded proteins homologous to enzymes involved in LOS biosynthesis: a heptosyl transferase (rfaF) involved in the synthesis of the LOS core and a ketodeoxyoctonate phosphate-dependent acyltransferase (htrB) that performs one of the late acylation reactions in lipid A synthesis. Inoculation of human bronchiolar xenografts revealed a significant reduction in colonization capacity by htrB mutants. In vitro, htrB mutants elicited lesser degrees of cytoskeletal rearrangement and less stimulation of host cell signaling with 16HBE14o(-) cells and decreased intracellular survival. These results implicate acylation of H. influenzae lipid A as playing a key role in the organisms' colonization of the normal airway.

Carbohydrate sulfation effects on growth of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a key player in the pathology and morbidity of cystic fibrosis. Chronic obstructive pulmonary disease, which results from the most common and severe mutations in this genetic disorder, typically includes chronic infection with P. aeruginosa which, even with rugged antibiotic and physical therapy regimens, is rarely eradicated. It is not known whether the increased oligosaccharide sulfation characteristic of cystic fibrosis tracheobronchial mucins plays a role in the survival of P. aeruginosa in the airway. In this study, sulfated monosaccharides were synthesized and tested for their effects on the growth of clinical isolates and laboratory strains of this organism when supplied as the sole carbon source in vitro. Carbohydrate sulfation was observed to reduce, but not prohibit, growth of P. aeruginosa on carbohydrates normally utilized in their nonsulfated form. The various sulfated sugars employed as the sole carbon source gave characteristic and consistent growth profiles and maximum growth values across the strains tested. P. aeruginosa isolates from patients with cystic fibrosis often express a mucoid phenotype, which is thought to contribute to their ability to survive in harsh conditions. Carbohydrate sulfation effects on growth did not differ significantly between mucoid and nonmucoid strains. These results suggest that the additional sulfation of tracheobronchial mucin documented in cystic fibrosis may in fact contribute to the mucin's resistance to utilization by P. aeruginosa and potentially other pathogens, providing an additional level of host protection, and limiting the available nutrient pool and thereby bacterial growth.