Gene-specific antibiotic development: Evaluating effectiveness and investigating antibiotic-resistant Escherichia coli mutants of phosphorodiamidate morpholino oligomers

Phosphorodiamidate morpholino oligomers (PMOs) are novel antisense drugs in the early stages of development. These synthetic DNA mimics contain the same bases found in DNA and anneal to RNA in a complementary fashion. PMOs have been designed that target genes responsible for producing essential proteins in organisms such as Escherichia coli. After the PMOs get into the bacterial cell (with peptides attached to facilitate their entry), they block translation of the targeted gene by annealing to mRNA and kill the cell. In this way, PMOs can be used as antibiotics. Tests with varying concentrations of drugs were completed both in vitro and in vivo (with mice) showing that PMOs can be as effective as ampicillin in stopping a bacterial infection of E. coli. This thesis will cover some of these tests, as well as experiments in which PMO-resistant mutant strains were discovered and isolated. Further work with several peptide-PMO conjugates showed that resistance was not a result of a change in the PMO target sequence and appeared to be peptide-related. Finally, out of two experiments designed to pinpoint genes required for E. coli to be susceptible to peptide-PMOs, a transposon knock-out experiment was successful in generating a PMO-resistant mutant and singling out nmpC as a gene of interest. Though this gene is identified as a pseudogene on the BLAST website, this research suggests that nmpC could have a function. Future research with this gene as well as repeating this method to find other genes of interest may increase knowledge of how PMOs work and aid in the design of more effective PMOs

Proteomic analyses of primary human villous trophoblasts exposed to flame retardant BDE-47 using SWATH-MS

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants and recognized developmental toxicants that are detectable in placental tissues. Higher levels of in utero PBDE exposure have been associated with an increased risk of adverse birth outcomes. During pregnancy, cytotrophoblasts (CTBs) from the placenta play critical roles in the formation of the maternal-fetal interface via uterine invasion and vascular remodeling. The differentiation of these cells towards an invasive phenotype is crucial for proper placental development. We previously have shown that BDE-47 can impact CTB viability and hinder the ability of these cells to migrate and invade. To expand on potential toxicological mechanisms, we utilized quantitative proteomic approaches to identify changes in the global proteome of mid-gestation primary human CTBs after exposure to BDE-47. Using sequential window acquisition of all theoretical fragment-ion spectra (SWATH), we identified 3024 proteins in our CTB model of differentiation/invasion. Over 200 proteins were impacted as a function of BDE-47 exposure (1 μM and 5 μM) across the treatment period (15, 24, and 39 h). The differentially expressed molecules displayed time- and concentration-dependent changes in expression and were enriched in pathways associated with aggregatory and adhesive processes. Network analysis identified CYFIP1, a molecule previously unexplored in a placental context, to be dysregulated at BDE-47 concentrations previously seen to impact CTB migration/invasion. Our SWATH-MS dataset thus demonstrates BDE-47 impacts the global proteome of differentiating CTBs and serves as a valuable resource for further understanding of the relationship between environmental chemical exposures and placental development and function. AVAILABILITY OF DATA AND MATERIAL: Raw chromatograms are deposited on the MassIVE proteomic database (https://massive.ucsd.edu) under accession number MSV000087870. Normalized relative abundances are also available as Table S1

Glycolytic and Non-glycolytic Functions of Mycobacterium tuberculosis Fructose-1,6-bisphosphate Aldolase, an Essential Enzyme Produced by Replicating and Non-replicating Bacilli

The search for antituberculosis drugs active against persistent bacilli has led to our interest in metallodependent class II fructose- 1,6-bisphosphate aldolase (FBA-tb), a key enzyme of gluconeogenesis absent from mammalian cells. Knock-out experiments at the fba-tb locus indicated that this gene is required for the growth of Mycobacterium tuberculosis on gluconeogenetic substrates and in glucose-containing medium. Surface labeling and enzymatic activity measurements revealed that this enzyme was exported to the cell surface of M. tuberculosis and produced under various axenic growth conditions including oxygen depletion and hence by non-replicating bacilli. Importantly, FBA-tb was also produced in vivo in the lungs of infected guinea pigs and mice. FBA-tb bound human plasmin(ogen) and protected FBA-tb-bound plasmin from regulation by α 2-antiplasmin, suggestive of an involvement of this enzyme in host/pathogen interactions. The crystal structures of FBA-tb in the native form and in complex with a hydroxamate substrate analog were determined to 2.35- and 1.9-Å resolution, respectively. Whereas inhibitor attachment had no effect on the plasminogen binding activity of FBA-tb, it competed with the natural substrate of the enzyme, fructose 1,6-bisphosphate, and substantiated a previously unknown reaction mechanism associated with metallodependent aldolases involving recruitment of the catalytic zinc ion by the substrate upon active site binding. Altogether, our results highlight the potential of FBA-tb as a novel therapeutic target against both replicating and non-replicating bacilli.Fil: Santangelo, María de la Paz. State University of Colorado - Fort Collins; Estados Unidos. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gest, Petra M.. State University of Colorado - Fort Collins; Estados UnidosFil: Guerin, Marcelo E.. Universidad del País Vasco; EspañaFil: Coinçon, Mathieu. University of Montreal; CanadáFil: Pham, Ha. State University of Colorado - Fort Collins; Estados UnidosFil: Ryan, Gavin. State University of Colorado - Fort Collins; Estados UnidosFil: Puckett, Susan E.. Cornell University; Estados UnidosFil: Spencer, John S.. State University of Colorado - Fort Collins; Estados UnidosFil: Gonzalez Juarrero, Mercedes. State University of Colorado - Fort Collins; Estados UnidosFil: Daher, Racha. Universite de Paris XI. Institut de Chimie Moléculaire et des Matériaux d'Orsay; FranciaFil: Lenaerts, Anne J.. State University of Colorado - Fort Collins; Estados UnidosFil: Schnappinger, Dirk. Cornell University; Estados UnidosFil: Therisod, Michel. Universite de Paris XI. Institut de Chimie Moléculaire et des Matériaux d'Orsay; FranciaFil: Ehrt, Sabine. Cornell University; Estados UnidosFil: Sygusch, Jurgen. University of Montreal; CanadáFil: Jackson, Mary. State University of Colorado - Fort Collins; Estados Unido

Bacterial Resistance to Antisense Peptide-Phosphorodiamidate Morpholino Oligomers

Peptide phosphorodiamidate morpholino oligomers (PPMO) are synthetic DNA mimics that bind complementary RNA and inhibit bacterial gene expression. (RFF)₃RXB- AcpP PPMO (R, arginine; F, phenylalanine; X, 6-aminohexanoic acid; B, β-alanine) is complementary to 11 bases of the essential gene acpP (encodes acyl carrier protein). The MIC of (RFF)₃RXB-AcpP was 2.5 μM (14 μg/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF)₃RXB-AcpP was 4 x 10⁻⁷ mutations/cell division. A spontaneous (RFF)₃RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)₃RXB-AcpP was 40 μM (224 μg/ml) in PR200.1. The MICs of standard antibiotics were identical in PR200.1 and W3110. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)₃RXB or peptides with a similar composition or pattern of cationic and non-polar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF)₃RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped to sbmA. Genetic complementation of PR200.1 or RR3 with sbmA restored susceptibility to (RFF)₃RXB-AcpP. Deletion of sbmA caused resistance to (RFF)₃RXB-AcpP. We conclude that resistance to (RFF)₃RXB-AcpP was linked to the peptide and not the PMO, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF)₃R-XB PPMOs may be transported across the plasma membrane by SbmA

Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy

To understand the molecular origins of diseases caused by ultraviolet and visible light, and also to develop photodynamic therapy, it is important to resolve the mechanism of photoinduced DNA damage. Damage to DNA bound to a photosensitizer molecule frequently proceeds by one-electron photo-oxidation of guanine, but the precise dynamics of this process are sensitive to the location and the orientation of the photosensitizer, which are very difficult to define in solution. To overcome this, ultrafast time-resolved infrared (TRIR) spectroscopy was performed on photoexcited ruthenium polypyridyl–DNA crystals, the atomic structure of which was determined by X-ray crystallography. By combining the X-ray and TRIR data we are able to define both the geometry of the reaction site and the rates of individual steps in a reversible photoinduced electron-transfer process. This allows us to propose an individual guanine as the reaction site and, intriguingly, reveals that the dynamics in the crystal state are quite similar to those observed in the solvent medium

Variations in Amino Acid Composition of Antisense Peptide-Phosphorodiamidate Morpholino Oligomer Affect Potency against Escherichia coli In Vitro and In Vivo ▿

The potency of antisense peptide-phosphorodiamidate morpholino oligomers (PPMOs) was improved by varying the peptide composition. An antisense phosphorodiamidate morpholino oligomer (PMO) complementary to the mRNA of the essential gene acpP (which encodes the acyl carrier protein required for lipid biosynthesis) in Escherichia coli was conjugated to the 5′ ends of various cationic membrane-penetrating peptides. Each peptide had one of three repeating sequence motifs: C-N-N (motif 1), C-N (motif 2), or C-N-C (motif 3), where C is a cationic residue and N is a nonpolar residue. Variations in the cationic residues included arginine, lysine, and ornithine (O). Variations in the nonpolar residues included phenylalanine, valine, β-alanine (B), and 6-aminohexanoic acid (X). The MICs of the PPMOs varied from 0.625 to >80 μM (about 3 to 480 μg/ml). Three of the most potent were the (RX)6B-, (RXR)4XB-, and (RFR)4XB-AcpP PMOs, which were further tested in mice infected with E. coli. The (RXR)4XB-AcpP PMO was the most potent of the three conjugates tested in mice. The administration of 30 μg (1.5 mg/kg of body weight) (RXR)4XB-AcpP PMO at 15 min postinfection reduced CFU/ml in blood by 102 to 103 within 2 to 12 h compared to the numbers in water-treated controls. All mice treated with 30 μg/dose of (RXR)4XB-AcpP PMO survived infection, whereas all water-treated mice died 12 h postinfection. The reduction in CFU/ml in blood was proportional to the dose of PPMO from 30 to 300 μg/ml. In summary, the C-N-C motif was more effective than the other two motifs, arginine was more effective than lysine or ornithine, phenylalanine was more effective than 6-aminohexanoic acid in vitro but not necessarily in vivo, and (RXR)4XB-AcpP PMO reduced bacterial infection and promoted survival at clinically relevant doses