Seth M. Daly

ω-Hydroxyemodin Limits Staphylococcus aureus Quorum Sensing-Mediated Pathogenesis and Inflammation

ABSTRACT Antibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing. Staphylococcus aureus is a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures of Penicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM prevented agr signaling by all four S. aureus agr alleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by the agr operon, preventing the interaction of AgrA with the agr P2 promoter. Importantly, OHM was efficacious in a mouse model of S. aureus SSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing of S. aureus in vitro in an agr-dependent manner. These data suggest that bacterial disarmament through the suppression of S. aureus QS may bolster the host innate immune response and limit inflammation.

Nox2 Modification of LDL Is Essential for Optimal Apolipoprotein B-mediated Control of agr Type III Staphylococcus aureus Quorum-sensing

Staphylococcus aureus contains an autoinducing quorum-sensing system encoded within the agr operon that coordinates expression of virulence genes required for invasive infection. Allelic variation within agr has generated four agr specific groups, agr I–IV, each of which secretes a distinct autoinducing peptide pheromone (AIP1-4) that drives agr signaling. Because agr signaling mediates a phenotypic change in this pathogen from an adherent colonizing phenotype to one associated with considerable tissue injury and invasiveness, we postulated that a significant contribution to host defense against tissue damaging and invasive infections could be provided by innate immune mechanisms that antagonize agr signaling. We determined whether two host defense factors that inhibit AIP1-induced agrI signaling, Nox2 and apolipoprotein B (apoB), also contribute to innate control of AIP3-induced agrIII signaling. We hypothesized that apoB and Nox2 would function differently against AIP3, which differs from AIP1 in amino acid sequence and length. Here we show that unlike AIP1, AIP3 is resistant to direct oxidant inactivation by Nox2 characteristic ROS. Rather, the contribution of Nox2 to defense against agrIII signaling is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against S. aureus agrIII infection by binding the secreted signaling peptide, AIP3, and preventing expression of the agr-driven virulence factors which mediate invasive infection. ApoB within the context of oxLDL also binds AIP 1-4 and oxLDL antagonizes agr signaling by all four agr alleles. Our results suggest that Nox2-mediated oxidation of LDL facilitates a conformational change in apoB to one sufficient for binding and sequestration of all four AIPs, demonstrating the interdependence of apoB and Nox2 in host defense against agr signaling. These data reveal a novel role for oxLDL in host defense against S. aureus quorum-sensing signaling.

Unprecedented C-2 arylation of indole with diazonium salts: Syntheses of 2,3-disubstituted indoles and their antimicrobial activity.

A novel reaction of indole with aryldiazonium salts leading to the formation of 2-aryl-3-(arylazo)indoles was discovered. The products were found to possess potent anti-MRSA and anti-LLVRE activities. The SAR studies indicate that the potentially metabolically labile azo functionality can be replaced with ether oxygen and thioether sulfur atoms without any loss of activity.

Identification of the Staphylococcus aureus vfrAB Operon, a Novel Virulence Factor Regulatory Locus

ABSTRACT During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis.

Development of a mimotope vaccine targeting the Staphylococcus aureus quorum sensing pathway.

A major hurdle in vaccine development is the difficulty in identifying relevant target epitopes and then presenting them to the immune system in a context that mimics their native conformation. We have engineered novel virus-like-particle (VLP) technology that is able to display complex libraries of random peptide sequences on a surface-exposed loop in the coat protein without disruption of protein folding or VLP assembly. This technology allows us to use the same VLP particle for both affinity selection and immunization, integrating the power of epitope discovery and epitope mimicry of traditional phage display with the high immunogenicity of VLPs. Previously, we showed that using affinity selection with our VLP platform identifies linear epitopes of monoclonal antibodies and subsequent immunization generates the proper antibody response. To test if our technology could identify immunologic mimotopes, we used affinity selection on a monoclonal antibody (AP4-24H11) that recognizes the Staphylococcus aureus autoinducing peptide 4 (AIP4). AIP4 is a secreted eight amino acid, cyclized peptide produced from the S. aureus accessory gene regulator (agrIV) quorum-sensing operon. The agr system coordinates density dependent changes in gene expression, leading to the upregulation of a host of virulence factors, and passive transfer of AP4-24H11 protects against S. aureus agrIV-dependent pathogenicity. In this report, we identified a set of peptides displayed on VLPs that bound with high specificity to AP4-24H11. Importantly, similar to passive transfer with AP4-24H11, immunization with a subset of these VLPs protected against pathogenicity in a mouse model of S. aureus dermonecrosis. These data are proof of principle that by performing affinity selection on neutralizing antibodies, our VLP technology can identify peptide mimics of non-linear epitopes and that these mimotope based VLP vaccines provide protection against pathogens in relevant animal models.

Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy

The lack of effective and well-tolerated therapies against antibiotic-resistant bacteria is a global public health problem leading to prolonged treatment and increased mortality. To improve the efficacy of existing antibiotic compounds, we introduce a new method for strategically inducing antibiotic hypersensitivity in pathogenic bacteria. Following the systematic verification that the AcrAB-TolC efflux system is one of the major determinants of the intrinsic antibiotic resistance levels in Escherichia coli, we have developed a short antisense oligomer designed to inhibit the expression of acrA and increase antibiotic susceptibility in E. coli. By employing this strategy, we can inhibit E. coli growth using 2- to 40-fold lower antibiotic doses, depending on the antibiotic compound utilized. The sensitizing effect of the antisense oligomer is highly specific to the targeted gene’s sequence, which is conserved in several bacterial genera, and the oligomer does not have any detectable toxicity against human cells. Finally, we demonstrate that antisense oligomers improve the efficacy of antibiotic combinations, allowing the combined use of even antagonistic antibiotic pairs that are typically not favored due to their reduced activities.

Inhibition of Pseudomonas aeruginosa by Peptide-conjugated Phosphorodiamidate Morpholino Oligomers

ABSTRACT Pseudomonas aeruginosa is a highly virulent, multidrug-resistant pathogen that causes significant morbidity and mortality in hospitalized patients and is particularly devastating in patients with cystic fibrosis. Increasing antibiotic resistance coupled with decreasing numbers of antibiotics in the developmental pipeline demands novel antibacterial approaches. Here, we tested peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which inhibit translation of complementary mRNA from specific, essential genes in P. aeruginosa. PPMOs targeted to acpP, lpxC, and rpsJ, inhibited P. aeruginosa growth in many clinical strains and activity of PPMOs could be enhanced 2- to 8-fold by the addition of polymyxin B nonapeptide at subinhibitory concentrations. The PPMO targeting acpP was also effective at preventing P. aeruginosa PAO1 biofilm formation and at reducing existing biofilms. Importantly, treatment with various combinations of a PPMO and a traditional antibiotic demonstrated synergistic growth inhibition, the most effective of which was the PPMO targeting rpsJ with tobramycin. Furthermore, treatment of P. aeruginosa PA103-infected mice with PPMOs targeting acpP, lpxC, or rpsJ significantly reduced the bacterial burden in the lungs at 24 h by almost 3 logs. Altogether, this study demonstrates that PPMOs targeting the essential genes acpP, lpxC, or rpsJ in P. aeruginosa are highly effective at inhibiting growth in vitro and in vivo. These data suggest that PPMOs alone or in combination with antibiotics represent a novel approach to addressing the problems associated with rapidly increasing antibiotic resistance in P. aeruginosa.

Peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) restores carbapenem susceptibility to NDM-1-positive pathogens in vitro and in vivo.

Objectives: The objective of this study was to test the efficacy of an inhibitor of the New Delhi metallo-&bgr;- lactamase (NDM-1). Inhibiting expression of this type of antibiotic-resistance gene has the potential to restore antibiotic susceptibility in all bacteria carrying the gene. Methods: We have constructed a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that selectively inhibits the expression of NDM-1 and examined its ability to restore susceptibility to meropenem in vitro and in vivo. Results: In vitro, the PPMO reduced the MIC of meropenem for three different genera of pathogens that express NDM-1. In a murine model of lethal E. coli sepsis, the PPMO improved survival (92%) and reduced systemic bacterial burden when given concomitantly with meropenem. Conclusions: These data show that a PPMO can restore antibiotic susceptibility in vitro and in vivo and that the combination of PPMO and meropenem may have therapeutic potential against certain class B carbapenem-resistant infections in multiple genera of Gram-negative pathogens.

MCR-1 Inhibition with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Restores Sensitivity to Polymyxin in Escherichia coli

ABSTRACT In late 2015, the first example of a transferrable polymyxin resistance mechanism in Gram-negative pathogens, MCR-1, was reported. Since that report, MCR-1 has been described to occur in many Gram-negative pathogens, and the mechanism of MCR-1-mediated resistance was rapidly determined: an ethanolamine is attached to lipid A phosphate groups, rendering the membrane more electropositive and repelling positively charged polymyxins. Acquisition of MCR-1 is clinically significant because polymyxins are frequently last-line antibiotics used to treat extensively resistant organisms, so acquisition of this mechanism might lead to pan-resistant strains. Therefore, the ability to inhibit MCR-1 and restore polymyxin sensitivity would be an important scientific advancement. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are antisense molecules that were designed to target mRNA, preventing translation. Peptide conjugation enhances cellular entry, but they are positively charged, so we tested our lead antibacterial PPMOs by targeting an essential Escherichia coli gene, acpP, and demonstrated that they were still effective in mcr-1-positive E. coli strains. We then designed and synthesized two PPMOs targeted to mcr-1 mRNA. Five clinical mcr-1-positive E. coli strains were resensitized to polymyxins by MCR-1 inhibition, reducing MICs 2- to 16-fold. Finally, therapeutic dosing of BALB/c mice with MCR-1 PPMO combined with colistin in a sepsis model reduced morbidity and bacterial burden in the spleen at 24 h and offered a survival advantage out to 5 days. This is the first example of a way to modulate colistin resistance with an antisense approach and may be a viable strategy to combat this globally emerging antibiotic resistance threat. IMPORTANCE Polymyxin use has been increasing as a last line of defense against Gram-negative pathogens with high-level resistance mechanisms, such as carbapenemases. The recently described MCR-1 is a plasmid-mediated mechanism of resistance to polymyxins. MCR-1 is currently found in Gram-negative organisms already possessing high-level resistance mechanisms, leaving clinicians few or no antibacterial options for infections caused by these strains. This study utilizes antisense molecules that target mRNA, preventing protein translation. Herein we describe antisense molecules that can be directly antibacterial because they target genes essential to bacterial growth or blockade of MCR-1, restoring polymyxin sensitivity. We also demonstrate that MCR-1 antisense molecules restore the efficacies of polymyxins in mouse models of E. coli septicemia. Considering all things together, we demonstrate that antisense molecules may be effective therapeutics either alone when they target an essential gene or combined with antibiotics when they target specific resistance mechanisms, such as those seen with MCR-1. IMPORTANCE Polymyxin use has been increasing as a last line of defense against Gram-negative pathogens with high-level resistance mechanisms, such as carbapenemases. The recently described MCR-1 is a plasmid-mediated mechanism of resistance to polymyxins. MCR-1 is currently found in Gram-negative organisms already possessing high-level resistance mechanisms, leaving clinicians few or no antibacterial options for infections caused by these strains. This study utilizes antisense molecules that target mRNA, preventing protein translation. Herein we describe antisense molecules that can be directly antibacterial because they target genes essential to bacterial growth or blockade of MCR-1, restoring polymyxin sensitivity. We also demonstrate that MCR-1 antisense molecules restore the efficacies of polymyxins in mouse models of E. coli septicemia. Considering all things together, we demonstrate that antisense molecules may be effective therapeutics either alone when they target an essential gene or combined with antibiotics when they target specific resistance mechanisms, such as those seen with MCR-1.

Employing high-frequency alternating magnetic fields for the non-invasive treatment of prosthetic joint infections

Treatment of prosthetic joint infection (PJI) usually requires surgical replacement of the infected joint and weeks of antibiotic therapy, due to the formation of biofilm. We introduce a non-invasive method for thermal destruction of biofilm on metallic implants using high-frequency (>100 kHz) alternating magnetic fields (AMF). In vitro investigations demonstrate a >5-log reduction in bacterial counts after 5 minutes of AMF exposure. Confocal and scanning electron microscopy confirm removal of biofilm matrix components within 1 minute of AMF exposure, and combination studies of antibiotics and AMF demonstrate a 5-log increase in the sensitivity of Pseudomonas aeruginosa to ciprofloxacin. Finite element analysis (FEA) simulations demonstrate that intermittent AMF exposures can achieve uniform surface heating of a prosthetic knee joint. In vivo studies confirm thermal damage is confined to a localized region (<2 mm) around the implant, and safety can be achieved using acoustic monitoring for the presence of surface boiling. These initial studies support the hypothesis that AMF exposures can eradicate biofilm on metal implants, and may enhance the effectiveness of conventional antibiotics.