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ABSTRACTTwo types of phosphorodiamidate morpholino oligomers (PMOs) were tested for inhibition of growth of Salmonella entericaserovar Typhimurium. Both PMOs have the same 11-base sequence that is antisense to the region near the start codon of acpP, which is essential for lipid biosynthesis and viability. To the 3′ end of each is attached the membrane-penetrating peptide (RXR)4XB (R, X, and B indicate arginine, 6-aminohexanoic acid, and β-alanine, respectively). One peptide-PMO (AcpP PPMO) has no charge on the PMO moiety. The second PPMO has three cations (piperazine) attached to the phosphorodiamidate linkages (3+Pip-AcpP PPMO). A scrambled-sequence PPMO (Scr PPMO) was synthesized for each type of PMO. The MICs of AcpP PPMO, 3+Pip-AcpP PPMO, and either one of the Scr PPMOs were 1.25 μM (7 μg/ml), 0.156 μM (0.94 μg/ml), and >160 μM (>900 μg/ml), respectively. 3+Pip-AcpP PPMO at 1.25 or 2.5 μM significantly reduced the growth rates of pure cultures, whereas AcpP PPMO or either Scr PPMO had no effect. However, the viable cell count was significantly reduced at either concentration of 3+Pip-AcpP PPMO or AcpP PPMO, but not with either Scr PPMO. In other experiments, macrophages were infected intracellularly with S. entericaand treated with 3 μM 3+Pip-AcpP PPMO. Intracellular bacteria were reduced >99% with 3+Pip-AcpP PPMO, whereas intracellular bacteria increased 3 orders of magnitude in untreated or Scr PPMO-treated cultures. We conclude that either AcpP PPMO or 3+Pip-AcpP PPMO inhibited growth of S. entericain pure culture and that 3+Pip-AcpP PPMO reduced intracellular viability of S. entericain macrophages.

ABSTRACTThe 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 coliwas 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 102to 103within 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.

ABSTRACTQuorum sensing (QS) is a widespread process in bacteria used to coordinate gene expression with cell density, diffusion dynamics, and spatial distribution through the production of diffusible chemical signals. To date, most studies on QS have focused on model bacteria that are amenable to genetic manipulation and capable of high growth rates, but many environmentally important bacteria have been overlooked. For example, representatives of proteobacteria that participate in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite, produce QS signals called acyl-homoserine lactones (AHLs). Nitrification emits nitrogen oxide gases (NO, NO2, and N2O), which are potentially hazardous compounds that contribute to global warming. Despite considerable interest in nitrification, the purpose of QS in the physiology/ecology of nitrifying bacteria is poorly understood. Through a quorum quenching approach, we investigated the role of QS in a well-studied AHL-producing nitrite oxidizer, Nitrobacter winogradskyi. We added a recombinant AiiA lactonase to N. winogradskyicultures to degrade AHLs to prevent their accumulation and to induce a QS-negative phenotype and then used mRNA sequencing (mRNA-Seq) to identify putative QS-controlled genes. Our transcriptome analysis showed that expression of nirKand nirKcluster genes (ncgABC) increased up to 19.9-fold under QS-proficient conditions (minus active lactonase). These data led to us to query if QS influenced nitrogen oxide gas fluxes in N. winogradskyi. Production and consumption of NOxincreased and production of N2O decreased under QS-proficient conditions. Quorum quenching transcriptome approaches have broad potential to identify QS-controlled genes and phenotypes in organisms that are not genetically tractable.IMPORTANCEBacterial cell-cell signaling, or quorum sensing (QS), is a method of bacterial communication and gene regulation that is well studied in bacteria. However, little is known about the purpose of QS in many environmentally important bacteria. Here, we demonstrate quorum quenching coupled with mRNA-Seq to identify QS-controlled genes and phenotypes in Nitrobacter winogradskyi, a nitrite-oxidizing bacterium. Nitrite oxidizers play an important role in the nitrogen cycle though their participation in nitrification, the aerobic oxidation of ammonia to nitrate via nitrite. Our quorum quenching approach revealed that QS influences production and consumption of environmentally important nitrogen oxide gases (NO, NO2, and N2O) in N. winogradskyi. This study demonstrated a novel technique for studying QS in difficult-to-work-with microorganisms and showed that nitrite oxidizers might also contribute to nitrification-dependent production of nitrogen oxide gases that contribute to global warming.