Antivirulence drugs disarm rather than kill pathogens and are thought to alleviate the problem of resistance, although there is no evidence to support this notion. Quorum sensing (QS) often controls cooperative virulence factor production and is therefore an attractive antivirulence target, for which inhibitors (QSI) have been developed. We designed a proof-of-principle experiment to investigate the impact of bacterial social interactions on the evolution of QSI resistance. We cocultured Pseudomonas aeruginosa QS-deficient mutants with small proportions of the QS-proficient wild type, which in the absence of QSI mimic QSI-sensitive and -resistant variants, respectively. We employed two different QS-dependent nutrients that are degraded by extracellular (public) and cell-associated (private) enzymes. QS mutants (QSI-sensitive mimics) behaved as social cheaters that delayed population growth and prevented enrichment of wild-type cooperators (QSI-resistant mimics) only when nutrient acquisition was public, suggesting that QSI resistance would not spread. This highlights the potential for antivirulence strategies that target cooperative behaviors and provides a conceptual framework for future studies.

Many bacteria possess cell density-dependent quorum-sensing (QS) systems that often regulate cooperative secretions involved in host-microbe or microbe-microbe interactions. These secretions, or "public goods," are frequently coregulated by stress and starvation responses. Here we provide a physiological rationale for such regulatory complexity in the opportunistic pathogen Pseudomonas aeruginosa. Using minimal-medium batch and chemostat cultures, we comprehensively characterized specific growth rate-limiting macronutrients as key triggers for the expression of extracellular enzymes and metabolites directly controlled by the las and rhl QS systems. Expression was unrelated to cell density, depended on the secreted product's elemental composition, and was induced only when the limiting nutrient was not also a building block of the product; rhl-dependent products showed the strongest response, caused by the largely las-independent induction of the regulator RhlR and its cognate signal. In agreement with the prominent role of the rhl system, slow growth inverted the las-to-rhl signal ratio, previously considered a characteristic distinguishing between planktonic and biofilm lifestyles. Our results highlight a supply-driven, metabolically prudent regulation of public goods that minimizes production costs and thereby helps stabilize cooperative behavior. Such regulation would be beneficial for QS-dependent public goods that act broadly and nonspecifically, and whose need cannot always be accurately assessed by the producing cell. Clear differences in the capacities of the las and rhl systems to integrate starvation signals help explain the existence of multiple QS systems in one cell.

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, ␤-alanine (B), and 6-aminohexanoic acid (X). The MICs of the PPMOs varied from 0.625 to >80 M (about 3 to 480 g/ml). Three of the most potent were the (RX) 6 B-, (RXR) 4 XB-, and (RFR) 4 XB-AcpP PMOs, which were further tested in mice infected with E. coli. The (RXR) 4 XB-AcpP PMO was the most potent of the three conjugates tested in mice. The administration of 30 g (1.5 mg/kg of body weight) (RXR) 4 XB-AcpP PMO at 15 min postinfection reduced CFU/ml in blood by 10 2 to 10 3 within 2 to 12 h compared to the numbers in water-treated controls. All mice treated with 30 g/dose of (RXR) 4 XB-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 g/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) 4 XB-AcpP PMO reduced bacterial infection and promoted survival at clinically relevant doses.

Background: Members of the Burkholderia cepacia complex (Bcc) cause significant morbidity and mortality in patients with chronic granulomatous disease (CGD) and cystic fibrosis (CF). Many Bcc strains are antibiotic resistant requiring the exploration of novel antimicrobial approaches including antisense technologies, such as phosphorodiamidate morpholino oligomers (PMOs). Methods: Peptide-conjugated PMOs (PPMOs) were developed to target the acpP gene, encoding an acyl carrier protein thought to be essential for growth. Their antimicrobial activities were tested against different strains of Bcc in vitro and in infection models. Results: PPMOs targeting acpP were bactericidal against clinical isolates of Bcc (> 4 log reduction), whereas a PPMO with a scrambled base sequence (Scr) had no effect on growth. Human neutrophils (PMN) were infected with B. multivorans, and treated with AcpP PPMO. AcpP PPMO augmented killing compared to PMN alone ± Scr PPMO. CGD mice infected with B. multivorans were treated with AcpP PPMO, Scr PPMO or water at 0, 3 and 6 hours post-infection. Compared to water treated controls, the AcpP PPMO treated mice showed a ~80% reduction in the risk of dying by day 30 and relatively little pathology. Conclusions: AcpP PPMO is active against Bcc infections in vitro and in vivo.

Nitrosomonas europaea and Nitrobacter winogradskyi were grown singly and in co-culture in chemostats to probe for physiological differences between the two growth conditions. Co-culture growth medium containing 60 mM NH4 (+) resulted in a cell density (0.20-0.29 OD600) greater than the sum of the densities in single chemostat cultures, i.e., 0.09-0.14 OD600 for N. europaea with 60 mM NH4 (+)and 0.04-0.06 OD600 for N. winogradskyi with 60 mM NO2 (-). The NO2 (-)- and NH4 (+)-dependent O2 uptake rates, qRT-PCR, and microscopic observations indicated that in co-culture, N. europaea contributed ~0.20 OD600 (~80 %) and N. winogradskyi ~0.05 OD600 (~20 %). In co-culture, the transcriptomes showed that the mRNA levels of 773 genes in N. europaea (30.2 % of the genes) and of 372 genes in N. winogradskyi (11.8 % of the genes) changed significantly. Total cell growth and the analysis of the transcriptome revealed that in co-culture, N. europaea benefits more than N. winogradskyi.

Both AcpP peptide-PMO and AcpP PMO significantly reduced bacteraemia and promoted survival of mice infected with E. coli W3110. The conjugate was about 50-100 times more potent than the PMO without attached peptide. The L-isomeric peptide-PMO was 10 times more potent than the D-isomeric equivalent. The conjugate apparently was toxic at doses > or = 2 x 300 microg/mouse (30 mg/kg). PMOs produced a sequence-specific antibiotic effect and the conjugate had a therapeutic index (toxic dose/effective dose) approximately equal to 10 in a mouse model of infection.

Cationic PMOs inhibited bacterial growth in vitro and in vivo, and Gux-PMOs were more effective than Pip-PMOs. However, neither was as effective as the equivalent PMO-peptide conjugates. Subcutaneous treatment showed that 5 + Gux-AcpP or (RXR)(4)XB-AcpP entered the circulatory system, reduced infection and increased survival.

cPeptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF) 3 RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is ␤-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential gene acpP (which encodes acyl carrier protein). The MIC of (RFF) 3 RXB-AcpP was 2.5 M (14 g/ml) in Escherichia coli W3110. The rate of spontaneous resistance of E. coli to (RFF) 3 RXB-AcpP was 4 ؋ 10 ؊7 mutations/cell division. A spontaneous (RFF) 3 RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF) 3 RXB-AcpP was 40 M (224 g/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence of acpP was identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF) 3 RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation in sbmA, which encodes an active transport protein. In separate experiments, a (RFF) 3 RXB-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) 3 RXB-AcpP. Deletion of sbmA caused resistance to (RFF) 3 RXB-AcpP. We conclude that resistance to (RFF) 3 RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations in sbmA. The data further suggest that (RFF) 3 R-XB PPMOs may be transported across the plasma membrane by SbmA.