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

The objective of this study was to test the efficacy of an inhibitor of the New Delhi metallo-β- 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

Apolipoprotein B Is an Innate Barrier against Invasive Staphylococcus aureus Infection

SummaryStaphylococcus aureus is both a colonizer of humans and a cause of severe invasive infections. Although the genetic basis for phenotype switching from colonizing to invasive has received significant study, knowledge of host factors that antagonize the switch is limited. We show that VLDL and LDL lipoproteins interfere with this switch by antagonizing the S. aureus agr quorum-sensing system that upregulates genes required for invasive infection. The mechanism of antagonism entails binding of the major structural protein of these lipoproteins, apolipoprotein B, to an S. aureus autoinducing pheromone, preventing attachment of this pheromone to the bacteria and subsequent signaling through its receptor, AgrC. Mice deficient in plasma apolipoprotein B, either genetically or pharmacologically, are more susceptible to invasive agr+ bacterial infection, but not to infection with an agr deletion mutant. Therefore, apolipoprotein B at homeostatic levels in blood is an essential innate defense effector against invasive S. aureus infection

Selective chemical inhibition of agr quorum sensing in Staphylococcus aureus promotes host defense with minimal impact on resistance.

Bacterial signaling systems are prime drug targets for combating the global health threat of antibiotic resistant bacterial infections including those caused by Staphylococcus aureus. S. aureus is the primary cause of acute bacterial skin and soft tissue infections (SSTIs) and the quorum sensing operon agr is causally associated with these. Whether efficacious chemical inhibitors of agr signaling can be developed that promote host defense against SSTIs while sparing the normal microbiota of the skin is unknown. In a high throughput screen, we identified a small molecule inhibitor (SMI), savirin (S. aureus virulence inhibitor) that disrupted agr-mediated quorum sensing in this pathogen but not in the important skin commensal Staphylococcus epidermidis. Mechanistic studies employing electrophoretic mobility shift assays and a novel AgrA activation reporter strain revealed the transcriptional regulator AgrA as the target of inhibition within the pathogen, preventing virulence gene upregulation. Consistent with its minimal impact on exponential phase growth, including skin microbiota members, savirin did not provoke stress responses or membrane dysfunction induced by conventional antibiotics as determined by transcriptional profiling and membrane potential and integrity studies. Importantly, savirin was efficacious in two murine skin infection models, abating tissue injury and selectively promoting clearance of agr+ but not Δagr bacteria when administered at the time of infection or delayed until maximal abscess development. The mechanism of enhanced host defense involved in part enhanced intracellular killing of agr+ but not Δagr in macrophages and by low pH. Notably, resistance or tolerance to savirin inhibition of agr was not observed after multiple passages either in vivo or in vitro where under the same conditions resistance to growth inhibition was induced after passage with conventional antibiotics. Therefore, chemical inhibitors can selectively target AgrA in S. aureus to promote host defense while sparing agr signaling in S. epidermidis and limiting resistance development

A tripartite cocktail of chimeric monoclonal antibodies passively protects mice against ricin, staphylococcal enterotoxin B and Clostridium perfringens epsilon toxin

Due to the fast-acting nature of ricin, staphylococcal enterotoxin B (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these agents are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge

A tripartite cocktail of chimeric monoclonal antibodies passively protects mice against ricin, staphylococcal enterotoxin B and Clostridium perfringens epsilon toxin

Due to the fast-acting nature of ricin, staphylococcal enterotoxin (SEB), and Clostridium perfringens epsilon toxin (ETX), it is necessary that therapeutic interventions following a bioterrorism incident by one of these toxins occur as soon as possible after intoxication. Moreover, because the clinical manifestations of intoxication by these toxins are likely to be indistinguishable from each other, especially following aerosol exposure, we have developed a cocktail of chimeric monoclonal antibodies that is capable of neutralizing all three toxins. The efficacy of this cocktail was demonstrated in mouse models of lethal dose toxin challenge

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

<div><p><em>Staphylococcus aureus</em> contains an autoinducing quorum-sensing system encoded within the <em>agr</em> operon that coordinates expression of virulence genes required for invasive infection. Allelic variation within <em>agr</em> has generated four <em>agr</em> specific groups, <em>agr</em> I–IV, each of which secretes a distinct autoinducing peptide pheromone (AIP1-4) that drives <em>agr</em> signaling. Because <em>agr</em> 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 <em>agr</em> signaling. We determined whether two host defense factors that inhibit AIP1-induced <em>agr</em>I signaling, Nox2 and apolipoprotein B (apoB), also contribute to innate control of AIP3-induced <em>agr</em>III 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 <em>agr</em>III signaling is through oxidation of LDL. ApoB in the context of oxLDL, and not LDL, provides optimal host defense against <em>S. aureus agr</em>III infection by binding the secreted signaling peptide, AIP3, and preventing expression of the <em>agr</em>-driven virulence factors which mediate invasive infection. ApoB within the context of oxLDL also binds AIP 1-4 and oxLDL antagonizes <em>agr</em> signaling by all four <em>agr</em> 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 <em>agr</em> signaling. These data reveal a novel role for oxLDL in host defense against <em>S. aureus</em> quorum-sensing signaling.</p> </div

Savirin inhibits AgrA-dependent transcription in clinical isolates.

<p>Effect of savirin (5 µg ml⁻¹) vs. vehicle on <i>psm alpha</i> transcripts determined by qRT-PCR in clinical <i>S. aureus</i> isolates of each <i>agr</i> allele after 5 hr of culture. Data are represented as the mean of 5 replicates. Significance determined by two-way repeated measures ANOVA.</p

Passage of <i>agr</i>+ LAC with savirin <i>in vivo</i> or <i>in vitro</i> does not induce resistance or tolerance to savirin inhibition of <i>agr</i> signaling.

<p>(<b>A</b> & <b>B</b>) In vivo passage of LAC sequentially through the skin of 10 individual mice for 24 hr in the presence of either 16 µg erythromycin and 0.12 µg clindamycin (<b>A</b>) or 5 µg savirin (<b>B</b>). (<b>A</b>) Percent survival after incubation overnight with 16 µg ml⁻¹ erythromycin and increasing concentrations of clindamycin of non-passaged and passaged LAC, mean ± SEM, n = 3. (<b>B</b>) Percent AIP1 induced fold change in RNAIII after incubation for 1 hr with increasing concentrations of savirin in non-passaged or passaged LAC, mean ± SEM, n = 4–5. (<b>C</b> & <b>D</b>) <i>In vitro</i> passage serially for 10 days of LAC with either 16 µg ml⁻¹ of erythromycin and 0.12 µg ml⁻¹ clindamycin (<b>C</b>) or 5 µg ml⁻¹ savirin (<b>D</b>). (<b>C</b>) Percent survival after incubation overnight with 16 µg ml−1 erythromycin and increasing concentrations of clindamycin of non-passaged and passaged LAC, mean ± SEM, n = 3. (<b>D</b>) Percent AIP1 induced fold change in RNAIII after 1 hr incubation with increasing concentrations of savirin in non-passaged and passaged LAC, mean ± SEM, n = 3. ***p<0.001 by two-tailed Student's <i>t</i>-test. (<b>E</b> & <b>F</b>) Assessment of savirin resistance at the colony level of LAC passaged <i>in vivo</i> with either antibiotics (E/C) or savirin. (<b>E</b>) In vivo passaged bacteria were plated on skim milk agar plates (diluted to give ∼15–20 colonies/plate) containing either vehicle or 10 µg ml⁻¹ savirin for 72 hr. Non-passaged LAC Δ<i>agr</i> is shown for comparison. Arrows are pointing to zones of proteolysis. The black bar is 5 mm. (<b>F</b>) Quantification of proteolytic and non-proteolytic colonies after 72 hr on milk agar plates containing either vehicle or savirin. Data are represented as mean ± SEM, n = 8 replicates of a representative experiment of two independent experiments.</p