Publications by authors named "Seth M Daly"

20 Publications

  • Page 1 of 1

Quantitative Hemolysis Assays.

Methods Mol Biol 2021 ;2341:25-30

Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, Kansas City, KS, USA.

Many strains of Staphylococcus aureus produce a variety of cytolysins that target many different cell types to both fight the immune system and acquire nutrients. This includes hemolysins which destroy erythrocytes and are well studied virulence factors. Traditionally, hemolysin activity is measured on blood agar plates due to the simplicity of the assay. While this is telling, it cannot encapsulate the full story because S. aureus is known to behave differently in broth and on agar. Furthermore, plate-based assays are primarily semiquantitative and often a more accurate determination of hemolytic potential is needed to discern differences between strains. Here, we describe a method to quantify hemolysin activity from broth or similarly grown cells.
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http://dx.doi.org/10.1007/978-1-0716-1550-8_4DOI Listing
August 2021

Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers Retain Activity against Multidrug-Resistant Pseudomonas aeruginosa and .

mBio 2021 01 12;12(1). Epub 2021 Jan 12.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

Most antimicrobials currently in the clinical pipeline are modifications of existing classes of antibiotics and are considered short-term solutions due to the emergence of resistance. represents a major challenge for new antimicrobial drug discovery due to its versatile lifestyle, ability to develop resistance to most antibiotic classes, and capacity to form robust biofilms on surfaces and in certain hosts such as those living with cystic fibrosis (CF). A precision antibiotic approach to treating could be achieved with an antisense method, specifically by using peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs). Here, we demonstrate that PPMOs targeting (acyl carrier protein), (UDP-(3--acyl)--acetylglucosamine deacetylase), and (30S ribosomal protein S10) inhibited the growth of several multidrug-resistant clinical isolates at levels equivalent to those that were effective against sensitive strains. Lead PPMOs reduced established pseudomonal biofilms alone or in combination with tobramycin or piperacillin-tazobactam. Lead PPMO dosing alone or combined with tobramycin in an acute pneumonia model reduced lung bacterial burden in treated mice at 24 h and reduced morbidity up to 5 days postinfection. PPMOs reduced bacterial burden of extensively drug-resistant in the same model and resulted in superior survival compared to conventional antibiotics. These data suggest that lead PPMOs alone or in combination with clinically relevant antibiotics represent a promising therapeutic approach for combating infections. Numerous Gram-negative bacteria are becoming increasingly resistant to multiple, if not all, classes of existing antibiotics. Multidrug-resistant bacteria are a major cause of health care-associated infections in a variety of clinical settings, endangering patients who are immunocompromised or those who suffer from chronic infections, such as people with cystic fibrosis (CF). Herein, we utilize antisense molecules that target mRNA of genes essential to bacterial growth, preventing the formation of the target proteins, including , , and We demonstrate here that antisense molecules targeted to essential genes, alone or in combination with clinically relevant antibiotics, were effective in reducing biofilms and protected mice in a lethal model of acute pneumonia.
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http://dx.doi.org/10.1128/mBio.02411-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844538PMC
January 2021

Complement Receptor 3 Contributes to the Sexual Dimorphism in Neutrophil Killing of .

J Immunol 2020 09 7;205(6):1593-1600. Epub 2020 Aug 7.

Department of Pharmaceutical Sciences, University of New Mexico College of Pharmacy, Albuquerque, NM 87131;

We previously reported sex differences in innate susceptibility to skin infection and that bone marrow neutrophils (BMN) from female mice have an enhanced ability to kill ex vivo compared with those of male mice. However, the mechanism(s) driving this sex bias in neutrophil killing have not been reported. Given the role of opsonins such as complement, as well as their receptors, in recognition and clearance, we investigated their contribution to the enhanced bactericidal capacity of female BMN. We found that levels of C3 in the serum and CR3 (CD11b/CD18) on the surface of BMN were higher in female compared with male mice. Consistent with increased CR3 expression following TNF-α priming, production of reactive oxygen species (ROS), an important bactericidal effector, was also increased in female versus male BMN in response to serum-opsonized Furthermore, blocking CD11b reduced both ROS levels and killing by murine BMN from both sexes. However, at the same concentration of CD11b blocking Ab, killing by female BMN was greatly reduced compared with those from male mice, suggesting CR3-dependent differences in bacterial killing between sexes. Overall, this work highlights the contributions of CR3, C3, and ROS to innate sex bias in the neutrophil response to Given that neutrophils are crucial for clearance, understanding the mechanism(s) driving the innate sex bias in neutrophil bactericidal capacity could identify novel host factors important for host defense against .
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http://dx.doi.org/10.4049/jimmunol.2000545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484027PMC
September 2020

Vaccination with VLPs Presenting a Linear Neutralizing Domain of Hla Elicits Protective Immunity.

Toxins (Basel) 2020 07 11;12(7). Epub 2020 Jul 11.

Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA.

The pore-forming cytotoxin α-hemolysin, or Hla, is a critical virulence factor that promotes infection by causing tissue damage, excessive inflammation, and lysis of both innate and adaptive immune cells, among other cellular targets. In this study, we asked whether a virus-like particle (VLP)-based vaccine targeting Hla could attenuate Hla-mediated pathogenesis. VLPs are versatile vaccine platforms that can be used to display target antigens in a multivalent array, typically resulting in the induction of high titer, long-lasting antibody responses. In the present study, we describe the first VLP-based vaccines that target Hla. Vaccination with either of two VLPs displaying a 21 amino-acid linear neutralizing domain (LND) of Hla protected both male and female mice from subcutaneous Hla challenge, evident by reduction in lesion size and neutrophil influx to the site of intoxication. Antibodies elicited by VLP-LND vaccination bound both the LND peptide and the native toxin, effectively neutralizing Hla and preventing toxin-mediated lysis of target cells. We anticipate these novel and promising vaccines being part of a multi-component vaccine to reduce severity of infection.
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http://dx.doi.org/10.3390/toxins12070450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404987PMC
July 2020

Staphylococcus aureus Fatty Acid Kinase FakA Modulates Pathogenesis during Skin Infection via Proteases.

Infect Immun 2020 07 21;88(8). Epub 2020 Jul 21.

Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas, USA

fatty acid kinase FakA is necessary for the incorporation of exogenous fatty acids into the lipid membrane. We previously demonstrated that the inactivation of leads to decreased α-hemolysin (Hla) production but increased expression of the proteases SspAB and aureolysin , and that the Δ mutant causes larger lesions than the wild type (WT) during murine skin infection. As expected, necrosis is Hla dependent in the presence or absence of FakA, as both and Δ mutants are unable to cause necrosis of the skin. At day 4 postinfection, while the Δ mutant maintains larger and more necrotic abscesses, bacterial numbers are similar to those of the WT, indicating the enhanced tissue damage of mice infected with the Δ mutant is not due to an increase in bacterial burden. At this early stage of infection, skin infected with the Δ mutant has decreased levels of proinflammatory cytokines, such as interleukin-17A (IL-17A) and IL-1α, compared to those of WT-infected skin. At a later stage of infection (day 7), abscess resolution and bacterial clearance are hindered in Δ mutant-infected mice. The paradoxical findings of decreased Hla but increased necrosis led us to investigate the role of the proteases regulated by FakA. Utilizing Δ and Δ mutants in both the WT and mutant backgrounds, we found that the absence of these proteases in a mutant reduced dermonecrosis to levels similar to those of the WT strain. These studies suggest that the overproduction of proteases is one factor contributing to the enhanced pathogenesis of the Δ mutant during skin infection.
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http://dx.doi.org/10.1128/IAI.00163-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375762PMC
July 2020

GPER activation protects against epithelial barrier disruption by Staphylococcus aureus α-toxin.

Sci Rep 2019 02 4;9(1):1343. Epub 2019 Feb 4.

University of New Mexico College of Pharmacy, Department of Pharmaceutical Sciences, Albuquerque, NM, 87131, USA.

Sex bias in innate defense against Staphylococcus aureus skin and soft tissue infection (SSTI) is dependent on both estrogen production by the host and S. aureus secretion of the virulence factor, α-hemolysin (Hla). The impact of estrogen signaling on the immune system is most often studied in terms of the nuclear estrogen receptors ERα and ERβ. However, the potential contribution of the G protein-coupled estrogen receptor (GPER) to innate defense against infectious disease, particularly with respect to skin infection, has not been addressed. Using a murine model of SSTI, we found that GPER activation with the highly selective agonist G-1 limits S. aureus SSTI and Hla-mediated pathogenesis, effects that were absent in GPER knockout mice. Specifically, G-1 reduced Hla-mediated skin lesion formation and pro-inflammatory cytokine production, while increasing bacterial clearance. In vitro, G-1 reduced surface expression of the Hla receptor, ADAM10, in a human keratinocyte cell line and increased resistance to Hla-mediated permeability barrier disruption. This novel role for GPER activation in skin innate defense against infectious disease suggests that G-1 may have clinical utility in patients with epithelial permeability barrier dysfunction or who are otherwise at increased risk of S. aureus infection, including those with atopic dermatitis or cancer.
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http://dx.doi.org/10.1038/s41598-018-37951-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362070PMC
February 2019

Morpholino oligomers tested in vitro, in biofilm and in vivo against multidrug-resistant Klebsiella pneumoniae.

J Antimicrob Chemother 2018 06;73(6):1611-1619

Department of Internal Medicine, 5323 Harry Hines Blvd., University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Background: Klebsiella pneumoniae is an opportunistic pathogen and many strains are multidrug resistant. KPC is one of the most problematic resistance mechanisms, as it confers resistance to most β-lactams, including carbapenems. A promising platform technology for treating infections caused by MDR pathogens is the nucleic acid-like synthetic oligomers that silence bacterial gene expression by an antisense mechanism.

Objectives: To test a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) in a mouse model of K. pneumoniae infection.

Methods: PPMOs were designed to target various essential genes of K. pneumoniae and screened in vitro against a panel of diverse strains. The most potent PPMOs were further tested for their bactericidal effects in broth cultures and in established biofilms. Finally, a PPMO was used to treat mice infected with a KPC-expressing strain.

Results: The most potent PPMOs targeted acpP, rpmB and ftsZ and had MIC75s of 0.5, 4 and 4 μM, respectively. AcpP PPMOs were bactericidal at 1-2 × MIC and reduced viable cells and biofilm mass in established biofilms. In a mouse pneumonia model, therapeutic intranasal treatment with ∼30 mg/kg AcpP PPMO improved survival by 89% and reduced bacterial burden in the lung by ∼3 logs. Survival was proportional to the dose of AcpP PPMO. Delaying treatment by 2, 8 or 24 h post-infection improved survival compared with control groups treated with PBS or scrambled sequence (Scr) PPMOs.

Conclusions: PPMOs have the potential to be effective therapeutic agents against KPC-expressing, MDR K. pneumoniae.
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http://dx.doi.org/10.1093/jac/dky058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6251509PMC
June 2018

Antisense Inhibitors Retain Activity in Pulmonary Models of Burkholderia Infection.

ACS Infect Dis 2018 05 1;4(5):806-814. Epub 2018 Mar 1.

Department of Internal Medicine , UT Southwestern , 5323 Harry Hines Blvd , Dallas , Texas 75390 , United States.

The Burkholderia cepacia complex is a group of Gram-negative bacteria that are opportunistic pathogens in immunocompromised individuals, such as those with cystic fibrosis (CF) or chronic granulomatous disease (CGD). Burkholderia are intrinsically resistant to many antibiotics and the lack of antibiotic development necessitates novel therapeutics. Peptide-conjugated phosphorodiamidate morpholino oligomers are antisense molecules that inhibit bacterial mRNA translation. Targeting of PPMOs to the gene acpP, which is essential for membrane synthesis, lead to defects in the membrane and ultimately bactericidal activity. Exploration of additional PPMO sequences identified the ATG and Shine-Dalgarno sites as the most efficacious for targeting acpP. The CF lung is a complex microenvironment, but PPMO inhibition was still efficacious in an artificial model of CF sputum. PPMOs had low toxicity in human CF cells at doses that were antibacterial. PPMOs also reduced the bacterial burden in the lungs of immunocompromised CyBB mice, a model of CGD. Finally, the use of multiple PPMOs was efficacious in inhibiting the growth of both Burkholderia and Pseudomonas in an in vitro model of coinfection. Due to the intrinsic resistance of Burkholderia to traditional antibiotics, PPMOs represent a novel and viable approach to the treatment of Burkholderia infections.
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http://dx.doi.org/10.1021/acsinfecdis.7b00235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669075PMC
May 2018

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

mBio 2017 11 7;8(6). Epub 2017 Nov 7.

Department of Internal Medicine, UT Southwestern Medical Center, Dallas, Texas, USA

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 gene, , and demonstrated that they were still effective in --positive strains. We then designed and synthesized two PPMOs targeted to mRNA. Five clinical -positive 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. 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 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.
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http://dx.doi.org/10.1128/mBio.01315-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5676038PMC
November 2017

Synthetic Antibacterial Peptide Exhibits Synergy with Oxacillin against MRSA.

ACS Med Chem Lett 2017 Aug 7;8(8):853-857. Epub 2017 Jul 7.

Biodesign Institute Center for Innovations in Medicine, Arizona State University, Tempe, Arizona 85281, United States.

One proposed solution to the crisis of antimicrobial resistant (AMR) infections is the development of molecules that potentiate the activity of antibiotics for AMR bacteria, such as methicillin-resistant (MRSA). Rather than develop broad spectrum compounds, we developed a peptide that could potentiate the activity of a narrow spectrum antibiotic, oxacillin. In this way, the combination treatment could narrowly target the resistant pathogen and limit impact on host flora. We developed a peptide, , composed of a binding peptide and a inhibitory peptide conjugated to a branched peptide scaffold, which has modest activity against but exhibits synergy with oxacillin for MRSA both and in a MRSA skin infection model. The low concentration of and sub-MIC concentration of oxacillin necessary for activity suggest that this molecule is a candidate for future medicinal chemistry optimization.
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http://dx.doi.org/10.1021/acsmedchemlett.7b00200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5554890PMC
August 2017

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

Sci Rep 2017 08 8;7(1):7520. Epub 2017 Aug 8.

Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX, USA.

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.
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http://dx.doi.org/10.1038/s41598-017-07321-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548742PMC
August 2017

VLP-based vaccine induces immune control of Staphylococcus aureus virulence regulation.

Sci Rep 2017 04 4;7(1):637. Epub 2017 Apr 4.

University of New Mexico College of Pharmacy, Department of Pharmaceutical Sciences, Albuquerque, NM, 87131, USA.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTIs) and mounting antibiotic resistance requires innovative treatment strategies. S. aureus uses secreted cyclic autoinducing peptides (AIPs) and the accessory gene regulator (agr) operon to coordinate expression of virulence factors required for invasive infection. Of the four agr alleles (agr types I-IV and corresponding AIPs1-4), agr type I isolates are most frequently associated with invasive infection. Cyclization via a thiolactone bond is essential for AIP function; therefore, recognition of the cyclic form of AIP1 may be necessary for antibody-mediated neutralization. However, the small sizes of AIPs and labile thiolactone bond have hindered vaccine development. To overcome this, we used a virus-like particle (VLP) vaccine platform (PP7) for conformationally-restricted presentation of a modified AIP1 amino acid sequence (AIP1S). Vaccination with PP7-AIP1S elicited AIP1-specific antibodies and limited agr-activation in vivo. Importantly, in a murine SSTI challenge model with a highly virulent agr type I S. aureus isolate, PP7-AIP1S vaccination reduced pathogenesis and increased bacterial clearance compared to controls, demonstrating vaccine efficacy. Given the contribution of MRSA agr type I isolates to human disease, vaccine targeting of AIP1-regulated virulence could have a major clinical impact in the fight against antibiotic resistance.
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http://dx.doi.org/10.1038/s41598-017-00753-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5429642PMC
April 2017

Inhibition of Bacterial Growth by Peptide-Conjugated Morpholino Oligomers.

Methods Mol Biol 2017 ;1565:115-122

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.

Morpholino oligomers (MOs) are antisense molecules designed for sequence-specific binding of target mRNA. In bacteria, inhibition is hypothesized to occur by preventing translation initiation. Cell-penetrating peptides may be conjugated to the 5'- or 3'-termini of an MO to enhance cellular entry and therefore inhibition. Here we describe the three standard microbiological assays to assess in vitro antibacterial MO efficacy.
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http://dx.doi.org/10.1007/978-1-4939-6817-6_10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497461PMC
February 2018

Inhibition of Pseudomonas aeruginosa by Peptide-Conjugated Phosphorodiamidate Morpholino Oligomers.

Antimicrob Agents Chemother 2017 04 24;61(4). Epub 2017 Mar 24.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas, USA

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 PPMOs targeted to , , and , inhibited 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 was also effective at preventing 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 with tobramycin. Furthermore, treatment of PA103-infected mice with PPMOs targeting , , or 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 , , or in are highly effective at inhibiting growth and 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 .
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http://dx.doi.org/10.1128/AAC.01938-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365667PMC
April 2017

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

J Antimicrob Chemother 2017 03;72(3):782-790

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Objectives: 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.
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http://dx.doi.org/10.1093/jac/dkw476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890718PMC
March 2017

Sequence-Specific Targeting of Bacterial Resistance Genes Increases Antibiotic Efficacy.

PLoS Biol 2016 09 15;14(9):e1002552. Epub 2016 Sep 15.

Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

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.
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http://dx.doi.org/10.1371/journal.pbio.1002552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025249PMC
September 2016

ω-Hydroxyemodin limits staphylococcus aureus quorum sensing-mediated pathogenesis and inflammation.

Antimicrob Agents Chemother 2015 Apr 2;59(4):2223-35. Epub 2015 Feb 2.

Department of Pharmaceutical Sciences, College of Pharmacy, University of New Mexico, Albuquerque, New Mexico, USA

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.
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http://dx.doi.org/10.1128/AAC.04564-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356798PMC
April 2015

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

PLoS One 2014 7;9(11):e111198. Epub 2014 Nov 7.

Department of Pharmaceutical Sciences, University of New Mexico School of Medicine, Albuquerque, NM United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111198PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224382PMC
July 2015

Identification of the Staphylococcus aureus vfrAB operon, a novel virulence factor regulatory locus.

Infect Immun 2014 May 18;82(5):1813-22. Epub 2014 Feb 18.

Center for Staphylococcal Research, Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

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.
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http://dx.doi.org/10.1128/IAI.01655-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3993444PMC
May 2014

Nox2 modification of LDL is essential for optimal apolipoprotein B-mediated control of agr type III Staphylococcus aureus quorum-sensing.

PLoS Pathog 2013 Feb 14;9(2):e1003166. Epub 2013 Feb 14.

Department of Pharmaceutical Sciences, University of New Mexico College of Pharmacy, Albuquerque, New Mexico, USA.

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.
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http://dx.doi.org/10.1371/journal.ppat.1003166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573103PMC
February 2013
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