Publications by authors named "Paul M Dunman"

92 Publications

Genomics of Staphylococcus aureus ocular isolates.

PLoS One 2021 3;16(5):e0250975. Epub 2021 May 3.

Department of Ophthalmology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America.

Staphylococcus aureus is a major cause of ocular infections, often resulting in devastating vision loss. Despite the significant morbidity associated with these infections, little is yet known regarding the specific strain types that may have a predilection for ocular tissues nor the set of virulence factors that drive its pathogenicity in this specific biological niche. Whole genome sequencing (WGS) can provide valuable insight in this regard by providing a prospective, comprehensive assessment of the strain types and virulence factors driving disease among specific subsets of clinical isolates. As such, a set of 163-member S. aureus ocular clinical strains were sequenced and assessed for both common strain types (multilocus sequence type (MLST), spa, agr) associated with ocular infections as well as the presence/absence of 235 known virulence factors in a high throughput manner. This ocular strain set was then directly compared to a fully sequenced 116-member non-ocular S. aureus strain set curated from NCBI in order to identify key differences between ocular and non-ocular S. aureus isolates. The most common sequence types found among ocular S. aureus isolates were ST5, ST8 and ST30, generally reflecting circulating non-ocular pathogenic S. aureus strains. However, importantly, ocular isolates were found to be significantly enriched for a set of enterotoxins, suggesting a potential role for this class of virulence factors in promoting ocular disease. Further genomic analysis revealed that these enterotoxins are located on mobile pathogenicity islands, thus horizontal gene transfer may promote the acquisition of enterotoxins, potentially amplifying S. aureus virulence in ocular tissues.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250975PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8092774PMC
May 2021

RnpA Inhibitors: Computational-Guided Design, Synthesis and Initial Biological Evaluation.

Antibiotics (Basel) 2021 Apr 14;10(4). Epub 2021 Apr 14.

Dipartimento di Scienze Farmaceutiche, Università degli Studi di Milano, Via Luigi Mangiagalli 25, 20133 Milano, Italy.

Antibiotic resistance is spreading worldwide and it has become one of the most important issues in modern medicine. In this context, the bacterial RNA degradation and processing machinery are essential processes for bacterial viability that may be exploited for antimicrobial therapy. In , RnpA has been hypothesized to be one of the main players in these mechanisms. RnpA is able to modulate mRNA degradation and complex with a ribozyme (), facilitating ptRNA maturation. Corresponding small molecule screening campaigns have recently identified a few classes of RnpA inhibitors, and their structure activity relationship (SAR) has only been partially explored. Accordingly, in the present work, using computational modeling of RnpA we identified putative crucial interactions of known RnpA inhibitors, and we used this information to design, synthesize, and biologically assess new potential RnpA inhibitors. The present results may be beneficial for the overall knowledge about RnpA inhibitors belonging to both RNPA2000-like thiosemicarbazides and JC-like piperidine carboxamides molecular classes. We evaluated the importance of the different key moieties, such as the dichlorophenyl and the piperidine of JC2, and the semithiocarbazide, the furan, and the -propylphenyl ring of RNPA2000. Our efforts could provide a foundation for further computational-guided investigations.
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http://dx.doi.org/10.3390/antibiotics10040438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071009PMC
April 2021

A Modular Synthetic Route Involving -Aryl-2-nitrosoaniline Intermediates Leads to a New Series of 3-Substituted Halogenated Phenazine Antibacterial Agents.

J Med Chem 2021 06 21;64(11):7275-7295. Epub 2021 Apr 21.

Department of Medicinal Chemistry, Center for Natural Products, Drug Discovery and Development (CNPD3), College of Pharmacy, University of Florida, Gainesville, Florida 32610, United States.

Pathogenic bacteria demonstrate incredible abilities to evade conventional antibiotics through the development of resistance and formation of dormant, surface-attached biofilms. Therefore, agents that target and eradicate planktonic and biofilm bacteria are of significant interest. We explored a new series of halogenated phenazines (HP) through the use of -aryl-2-nitrosoaniline synthetic intermediates that enabled functionalization of the 3-position of this scaffold. Several HPs demonstrated potent antibacterial and biofilm-killing activities (, HP , against methicillin-resistant : MIC = 0.075 μM; MBEC = 2.35 μM), and transcriptional analysis revealed that HPs , , and induce rapid iron starvation in MRSA biofilms. Several HPs demonstrated excellent activities against (HP , MIC = 0.80 μM against CDC1551). This work established new SAR insights, and HP demonstrated efficacy in dorsal wound infection models in mice. Encouraged by these findings, we believe that HPs could lead to significant advances in the treatment of challenging infections.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00168DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192493PMC
June 2021

Optimization of 2-Acylaminocycloalkylthiophene Derivatives for Activity against RnpA.

Antibiotics (Basel) 2021 Mar 31;10(4). Epub 2021 Mar 31.

Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642, USA.

is well-recognized to cause debilitating bacterial infections that are difficult to treat due to the emergence of antibiotic resistance. As such, there is a need to develop new antimicrobials for the therapeutic intervention of disease. To that end, RnpA is an essential enzyme that is hypothesized to participate in two required cellular processes, precursor tRNA (ptRNA) maturation and mRNA degradation. Corresponding high throughput screening campaigns have identified the phenylcarbamoyl cyclic thiopenes as a chemical class of RnpA inhibitors that display promising antibacterial effects by reducing RnpA ptRNA and mRNA degradation activities and low human cell toxicity. Herein, we perform a structure activity relationship study of the chemical scaffold. Results revealed that the cycloalkane ring size and trifluoroacetamide moiety are required for antibacterial activity, whereas modifications of the para and/or meta positions of the pharmacophore's phenyl group allowed tuning of the scaffold's antimicrobial performance and RnpA inhibitory activity. The top performing compounds with respect to antimicrobial activity also did not exhibit cytotoxicity to human cell lines at concentrations up to 100 µM, greater than 100-fold the minimum inhibitory concentration (MIC). Focused studies of one analog, RNP0012, which exhibited the most potent antimicrobial and inhibition of cellular RnpA activities revealed that the compound reduced bacterial burden in a murine model of disease. Taken together, the results presented are expected to provide an early framework for optimization of next-generation of RnpA inhibitor analogues that may represent progenitors of a new class of antimicrobials.
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http://dx.doi.org/10.3390/antibiotics10040369DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066339PMC
March 2021

Staphylococcus aureus resistance to albocycline can be achieved by mutations that alter cellular NAD/PH pools.

Bioorg Med Chem 2021 02 7;32:115995. Epub 2021 Jan 7.

Department of Chemistry, Temple University, Philadelphia, PA 19122, United States. Electronic address:

Small molecule target identification is a critical step in modern antibacterial drug discovery, particularly against multi-drug resistant pathogens. Albocycline (ALB) is a macrolactone natural product with potent activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) whose mechanism of action has been elusive to date. Herein, we report biochemical and genomic studies that reveal ALB does not target bacterial peptidoglycan biosynthesis or the ribosome; rather, it appears to modulate NADPH ratios and upregulate redox sensing in the cell consistent with previous studies at Upjohn. Owing to the complexity inherent in biological pathways, further genomic assays are needed to identify the true molecular target(s) of albocycline.
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http://dx.doi.org/10.1016/j.bmc.2021.115995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891091PMC
February 2021

Synthesis and biological evaluation of semi-synthetic albocycline analogs.

Bioorg Med Chem Lett 2020 11 19;30(21):127509. Epub 2020 Aug 19.

Department of Chemistry, Temple University, Philadelphia, PA 19122, United States. Electronic address:

Albocycline (ALB) is a unique macrolactone natural product with potent, narrow-spectrum activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate (VISA), and vancomycin-resistant S. aureus (VRSA) strains (MIC = 0.5-1.0 μg/mL). Described herein is the synthesis and evaluation of a novel series analogs derived from albocycline by functionalization at three specific sites: the C2-C3 enone, the tertiary carbinol at C4, and the allylic C16 methyl group. Exploration of the structure-activity relationships (SAR) by means of minimum inhibitory concentration assays (MICs) revealed that C4 ester analog 6 was twice as potent as ALB, which represents a class of lead compound that can be further studied to address multi-drug resistant pathogens.
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http://dx.doi.org/10.1016/j.bmcl.2020.127509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7577960PMC
November 2020

Optimization of 4-Substituted Benzenesulfonamide Scaffold To Reverse Acinetobacter baumannii Serum-Adaptive Efflux Associated Antibiotic Tolerance.

ChemMedChem 2020 09 13;15(18):1731-1740. Epub 2020 Aug 13.

Department of Medicinal Chemistry and Molecular Pharmacology College of Pharmacy, Purdue University, 575 Stadium Mall Dr., West Lafayette, IN, 47907.

Acinetobacter baumannii is a nosocomial pathogen of urgent concern for public health due to rising rates of multidrug and pandrug resistance. In the context of environmental cues such as growth in human serum, A. baumannii is known to display adaptive efflux, in which a multitude of efflux-associated genes are upregulated, resulting in efflux-mediated drug tolerance in strains that are otherwise susceptible to antibiotic therapy. Previously, we identified a sulfonamide-containing scaffold molecule (ABEPI1) that reversed serum-associated antibiotic tolerance in A. baumannii. Herein, we present structure-activity relationship studies on 29 newly synthesized analogues. These molecules were characterized for their ability to potentiate multiple antibiotics in serum, reduce serum-associated ethidium bromide efflux and depolarize bacterial cell membranes. In addition, they were assessed for toxicity to mammalian cells. Collectively, these molecules may represent promising potential adjuvants for use in combination with new and existing antibiotics to treat A. baumannii bacterial infections.
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http://dx.doi.org/10.1002/cmdc.202000328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899150PMC
September 2020

Serum-Associated Antibiotic Tolerance in Pediatric Clinical Isolates of Pseudomonas aeruginosa.

J Pediatric Infect Dis Soc 2020 Dec;9(6):671-679

Department of Medicine, Division of Infectious Diseases, Johns Hopkins All Children's Hospital, St. Petersburg, Florida, USA.

Background: When grown in human serum, laboratory isolates of Pseudomonas aeruginosa exhibit tolerance to antibiotics at inhibitory concentrations. This phenomenon, known as serum-associated antibiotic tolerance (SAT), could lead to clinical treatment failure of pseudomonal infections. Our purpose in this study was to determine the prevalence and clinical impact of SAT in Pseudomonas isolates in hospitalized children.

Methods: The SAT phenotype was assessed in patients aged <18 years admitted with respiratory or blood cultures positive for P. aeruginosa. The SAT phenotype was a priori defined as a ≥2-log increase in colony-forming units when grown in human serum compared with Luria-Bertani medium in the presence of minocycline or tobramycin.

Results: SAT was detected in 29 (64%) patients. Fourteen patients each (34%) had cystic fibrosis (CF) and tracheostomies. Patient demographics and comorbidities did not differ by SAT status. Among CF patients, SAT was associated with longer duration of intravenous antibiotics (10 days vs 5 days; P < .01).

Conclusions: This study establishes that SAT exists in P. aeruginosa from human serum and may be a novel factor that contributes to differences in clinical outcomes. Future research should investigate the mechanisms that contribute to SAT in order to identify novel targets for adjunctive antimicrobial therapies.
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http://dx.doi.org/10.1093/jpids/piz094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7974018PMC
December 2020

A Novel, Broad-Spectrum Antimicrobial Combination for the Treatment of Pseudomonas aeruginosa Corneal Infections.

Antimicrob Agents Chemother 2019 10 23;63(10). Epub 2019 Sep 23.

Department of Ophthalmology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

Bacterial keratitis causes significant blindness, yet antimicrobial resistance has rendered current treatments ineffective. Polymyxin B-trimethoprim (PT) plus rifampin has potent activity against and , two important causes of keratitis. Here we further characterize this combination against in a murine keratitis model. PT plus rifampin performed comparably to or better than moxifloxacin, the gold standard, suggesting that the combination may be a promising therapy for bacterial keratitis.
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http://dx.doi.org/10.1128/AAC.00777-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761539PMC
October 2019

Characterizing the Antimicrobial Properties of 405 nm Light and the Corning® Light-Diffusing Fiber Delivery System.

Lasers Surg Med 2019 12 14;51(10):887-896. Epub 2019 Jul 14.

Division of Science and Technology, Corning Research & Development Corporation, Corning, New York, 14831.

Background And Objectives: Hospital-acquired infections (HAIs) and multidrug resistant bacteria pose a significant threat to the U.S. healthcare system. With a dearth of new antibiotic approvals, novel antimicrobial strategies are required to help solve this problem. Violet-blue visible light (400-470 nm) has been shown to elicit strong antimicrobial effects toward many pathogens, including representatives of the ESKAPE bacterial pathogens, which have a high propensity to cause HAIs. However, phototherapeutic solutions to prevention or treating infections are currently limited by efficient and nonobtrusive light-delivery mechanisms.

Study Design/materials And Methods: Here, we investigate the in vitro antimicrobial properties of flexible Corning® light-diffusing fiber (LDF) toward members of the ESKAPE pathogens in a variety of growth states and in the context of biological materials. Bacteria were grown on agar surfaces, in liquid culture and on abiotic surfaces. We also explored the effects of 405 nm light within the presence of lung surfactant, human serum, and on eukaryotic cells. Pathogens tested include Enterococcus spp, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., Staphylococcus epidermidis, Streptococcus pyogenes, Candida albicans, and Escherichia coli.

Results: Overall, the LDF delivery of 405 nm violet-blue light exerted a significant degree of microbicidal activity against a wide range of pathogens under diverse experimental conditions.

Conclusions: The results exemplify the fiber's promise as a non-traditional approach for the prevention and/or therapeutic intervention of HAIs. Lasers Surg. Med. © 2019 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/lsm.23132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916415PMC
December 2019

Identification of Small Molecule Inhibitors of RnpA.

Antibiotics (Basel) 2019 Apr 28;8(2). Epub 2019 Apr 28.

Department of Microbiology and Immunology, University of Rochester School of Medicine, Rochester, NY 14642, USA.

RnpA is thought to be a unique dual functional antimicrobial target that is required for two essential cellular processes, precursor tRNA processing and messenger RNA degradation. Herein, we used a previously described whole cell-based mupirocin synergy assay to screen members of a 53,000 compound small molecule diversity library and simultaneously enrich for agents with cellular RnpA inhibitory activity. A medicinal chemistry-based campaign was launched to generate a preliminary structure activity relationship and guide early optimization of two novel chemical classes of RnpA inhibitors identified, phenylcarbamoyl cyclic thiophene and piperidinecarboxamide. Representatives of each chemical class displayed potent anti-staphylococcal activity, limited the protein's in vitro ptRNA processing and mRNA degradation activities, and exhibited favorable therapeutic indexes. The most potent piperidinecarboxamide RnpA inhibitor, JC2, displayed inhibition of cellular RnpA mRNA turnover, RnpA-depletion strain hypersusceptibility, and exhibited antimicrobial efficacy in a wax worm model of infection. Taken together, these results establish that the whole cell screening assay used is amenable to identifying small molecule RnpA inhibitors within large chemical libraries and that the chemical classes identified here may represent progenitors of new classes of antimicrobials that target RnpA.
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http://dx.doi.org/10.3390/antibiotics8020048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627331PMC
April 2019

Calcium Phosphate Spacers for the Local Delivery of Sitafloxacin and Rifampin to Treat Orthopedic Infections: Efficacy and Proof of Concept in a Mouse Model of Single-Stage Revision of Device-Associated Osteomyelitis.

Pharmaceutics 2019 Feb 22;11(2). Epub 2019 Feb 22.

Department of Biomedical Engineering, University of Rochester, Rochester, NY 14642, USA.

Osteomyelitis is a chronic bone infection that is often treated with adjuvant antibiotic-impregnated poly(methyl methacrylate) (PMMA) cement spacers in multi-staged revisions. However, failure rates remain substantial due to recurrence of infection, which is attributed to the poor performance of the PMMA cement as a drug release device. Hence, the objective of this study was to design and evaluate a bioresorbable calcium phosphate scaffold (CaPS) for sustained antimicrobial drug release and investigate its efficacy in a murine model of femoral implant-associated osteomyelitis. Incorporating rifampin and sitafloxacin, which are effective against bacterial phenotypes responsible for bacterial persistence, into 3D-printed CaPS coated with poly(lactic co-glycolic) acid, achieved controlled release for up to two weeks. Implantation into the murine infection model resulted in decreased bacterial colonization rates at 3- and 10-weeks post-revision for the 3D printed CaPS in comparison to gentamicin-laden PMMA. Furthermore, a significant increase in bone formation was observed for 3D printed CaPS incorporated with rifampin at 3 and 10 weeks. The results of this study demonstrate that osteoconductive 3D printed CaPS incorporated with antimicrobials demonstrate more efficacious bacterial colonization outcomes and bone growth in a single-stage revision in comparison to gentamicin-laden PMMA requiring a two-stage revision.
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http://dx.doi.org/10.3390/pharmaceutics11020094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6410209PMC
February 2019

Development of a Broad-Spectrum Antimicrobial Combination for the Treatment of Staphylococcus aureus and Pseudomonas aeruginosa Corneal Infections.

Antimicrob Agents Chemother 2019 01 21;63(1). Epub 2018 Dec 21.

Department of Ophthalmology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

and are two of the most common causes of bacterial keratitis and corresponding corneal blindness. Accordingly, such infections are predominantly treated with broad-spectrum fluoroquinolones, such as moxifloxacin. Yet, the rising fluoroquinolone resistance has necessitated the development of alternative therapeutic options. Herein, we describe the development of a polymyxin B-trimethoprim (PT) ophthalmic formulation containing the antibiotic rifampin, which exhibits synergistic antimicrobial activity toward a panel of contemporary ocular clinical and isolates, low spontaneous resistance frequency, and bactericidal kinetics and antibiofilm activities equaling or exceeding the antimicrobial properties of moxifloxacin. The PT plus rifampin combination also demonstrated increased efficacy in comparison to those of either commercial PT or moxifloxacin in a murine keratitis model of infection, resulting in bacterial clearance of 70% in the animals treated. These results suggest that the combination of PT and rifampin may represent a novel antimicrobial agent in the treatment of bacterial keratitis.
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http://dx.doi.org/10.1128/AAC.01929-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325218PMC
January 2019

A High-Throughput Screening Approach To Repurpose FDA-Approved Drugs for Bactericidal Applications against Staphylococcus aureus Small-Colony Variants.

mSphere 2018 10 31;3(5). Epub 2018 Oct 31.

Department of Biomedical Engineering, University of Rochester, Rochester, New York, USA.

Drug repurposing offers an expedited and economical route to develop new clinical therapeutics in comparison to traditional drug development. Growth-based high-throughput screening is concomitant with drug repurposing and enables rapid identification of new therapeutic uses for investigated drugs; however, this traditional method is not compatible with microorganisms with abnormal growth patterns such as small-colony variants (SCV). SCV subpopulations are auxotrophic for key compounds in biosynthetic pathways, which result in low growth rate. SCV formation is also associated with reduced antibiotic susceptibility, and the SCV's ability to revert to the normal cell growth state is thought to contribute to recurrence of infections. Thus, there is a critical need to identify antimicrobial agents that are potent against SCV in order to effectively treat chronic infections. Accordingly, here we describe adapting an adenylate kinase (AK)-based cell death reporter assay to identify members of a Food and Drug Administration (FDA)-approved drug library that display bactericidal activity against SCV. Four library members, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, exhibited potent SCV bactericidal activity against a stable SCV. Further investigation showed that sitafloxacin was potent against methicillin-susceptible and -resistant , as well as within an established biofilm. Taken together, these results demonstrate the ability to use the AK assay to screen small-molecule libraries for SCV bactericidal agents and highlight the therapeutic potential of sitafloxacin to be repurposed to treat chronic infections associated with SCV and/or biofilm growth states. Conventional antibiotics fail to successfully treat chronic osteomyelitis, endocarditis, and device-related and airway infections. These recurring infections are associated with the emergence of SCV, which are recalcitrant to conventional antibiotics. Studies have investigated antibiotic therapies to treat SCV-related infections but have had little success, emphasizing the need to identify novel antimicrobial drugs. However, drug discovery is a costly and time-consuming process. An alternative strategy is drug repurposing, which could identify FDA-approved and well-characterized drugs that could have off-label utility in treating SCV. In this study, we adapted a high-throughput AK-based assay to identify 4 FDA-approved drugs, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, which display antimicrobial activity against SCV, suggesting an avenue for drug repurposing in order to effectively treat SCV-related infections. Additionally, this screening paradigm can easily be adapted for other drug/chemical libraries to identify compounds bactericidal against SCV.
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http://dx.doi.org/10.1128/mSphere.00422-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6211227PMC
October 2018

Crystal structure of the ribonuclease-P-protein subunit from Staphylococcus aureus.

Acta Crystallogr F Struct Biol Commun 2018 Oct 19;74(Pt 10):632-637. Epub 2018 Sep 19.

Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, IN 47907, USA.

Staphylococcus aureus ribonuclease-P-protein subunit (RnpA) is a promising antimicrobial target that is a key protein component for two essential cellular processes, RNA degradation and transfer-RNA (tRNA) maturation. The first crystal structure of RnpA from the pathogenic bacterial species, S. aureus, is reported at 2.0 Å resolution. The structure presented maintains key similarities with previously reported RnpA structures from bacteria and archaea, including the highly conserved RNR-box region and aromatic residues in the precursor-tRNA 5'-leader-binding domain. This structure will be instrumental in the pursuit of structure-based designed inhibitors targeting RnpA-mediated RNA processing as a novel therapeutic approach for treating S. aureus infections.
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http://dx.doi.org/10.1107/S2053230X18011512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168776PMC
October 2018

Elucidating the inhibition of peptidoglycan biosynthesis in Staphylococcus aureus by albocycline, a macrolactone isolated from Streptomyces maizeus.

Bioorg Med Chem 2018 07 24;26(12):3453-3460. Epub 2018 May 24.

Department of Chemistry, Temple University, Philadelphia, PA 19122, United States. Electronic address:

Antibiotic resistance is a serious threat to global public health, and methicillin-resistant Staphylococcus aureus (MRSA) is a poignant example. The macrolactone natural product albocycline, derived from various Streptomyces strains, was recently identified as a promising antibiotic candidate for the treatment of both MRSA and vancomycin-resistant S. aureus (VRSA), which is another clinically relevant and antibiotic resistant strain. Moreover, it was hypothesized that albocycline's antimicrobial activity was derived from the inhibition of peptidoglycan (i.e., bacterial cell wall) biosynthesis. Herein, preliminary mechanistic studies are performed to test the hypothesis that albocycline inhibits MurA, the enzyme that catalyzes the first step of peptidoglycan biosynthesis, using a combination of biological assays alongside molecular modeling and simulation studies. Computational modeling suggests albocycline exists as two conformations in solution, and computational docking of these conformations to an ensemble of simulated receptor structures correctly predicted preferential binding to S. aureus MurA-the enzyme that catalyzes the first step of peptidoglycan biosynthesis-over Escherichia coli (E. coli) MurA. Albocycline isolated from the producing organism (Streptomyces maizeus) weakly inhibited S. aureus MurA (IC of 480 μM) but did not inhibit E. coli MurA. The antimicrobial activity of albocycline against resistant S. aureus strains was superior to that of vancomycin, preferentially inhibiting Gram-positive organisms. Albocycline was not toxic to human HepG2 cells in MTT assays. While these studies demonstrate that albocycline is a promising lead candidate against resistant S. aureus, taken together they suggest that MurA is not the primary target, and further work is necessary to identify the major biological target.
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http://dx.doi.org/10.1016/j.bmc.2018.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6008248PMC
July 2018

Endoribonuclease YbeY Is Linked to Proper Cellular Morphology and Virulence in Brucella abortus.

J Bacteriol 2018 06 24;200(12). Epub 2018 May 24.

Department of Biomedical Sciences and Pathobiology, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA

The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in is linked to a variety of important activities, including proper cellular morphology, mRNA transcript levels, and virulence. Deletion of in led to a small-colony phenotype when the bacteria were grown on agar medium, as well as to significant aberrations in the morphology of the bacterial cell as evidenced by electron microscopy. Additionally, compared to the parental strain, the Δ strain was significantly attenuated in both macrophage and mouse models of infection. The Δ strain also showed increased sensitivities to several -applied stressors, including bile acid, hydrogen peroxide, SDS, and paraquat. Transcriptomic analysis revealed that a multitude of mRNA transcripts are dysregulated in the Δ strain, and many of the identified mRNAs encode proteins involved in metabolism, nutrient transport, transcriptional regulation, and flagellum synthesis. We subsequently constructed gene deletion strains of the most highly dysregulated systems, and several of the YbeY-linked gene deletion strains exhibited defects in the ability of the bacteria to survive and replicate in primary murine macrophages. Taken together, these data establish a clear role for YbeY in the biology and virulence of ; moreover, this work further illuminates the highly varied roles of this widely conserved endoribonuclease in bacteria. spp. are highly efficient bacterial pathogens of animals and humans, causing significant morbidity and economic loss worldwide, and relapse of disease often occurs following antibiotic treatment of human brucellosis. As such, novel therapeutic strategies to combat infections are needed. Ribonucleases in the brucellae are understudied, and these enzymes represent elements that may be potential targets for future treatment approaches. The present work demonstrates the importance of the YbeY endoribonuclease for cellular morphology, efficient control of mRNA levels, and virulence in Overall, the results of this study advance our understanding of the critical roles of YbeY in the pathogenesis of the intracellular brucellae and expand our understanding of this highly conserved RNase.
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http://dx.doi.org/10.1128/JB.00105-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971472PMC
June 2018

Novel inhibitors of Staphylococcus aureus RnpA that synergize with mupirocin.

Bioorg Med Chem Lett 2018 04 31;28(6):1127-1131. Epub 2018 Jan 31.

Moulder Center for Drug Discovery Research, Temple University School of Pharmacy, 3307 N. Broad Street, Philadelphia, PA, United States. Electronic address:

We recently discovered RnpA as a promising new drug discovery target for methicillin-resistant S. aureus (MRSA). RnpA is an essential protein that is thought to perform two required cellular processes. As part of the RNA degrasome Rnpa mediates RNA degradation. In combination with rnpB it forms RNase P haloenzymes which are required for tRNA maturation. A high throughput screen identified RNPA2000 as an inhibitor of both RnpA-associated activities that displayed antibacterial activity against clinically relevant strains of S. aureus, including MRSA. Structure-activity studies aimed at improving potency and replacing the potentially metabotoxic furan moiety led to the identification of a number of more potent analogs. Many of these new analogs possessed overt cellular toxicity that precluded their use as antibiotics but two derivatives, including compound 5o, displayed an impressive synergy with mupirocin, an antibiotic used for decolonizing MSRA whose effectiveness has recently been jeopardized by bacterial resistance. Based on our results, compounds like 5o may ultimately find use in resensitizing mupirocin-resistant bacteria to mupirocin.
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http://dx.doi.org/10.1016/j.bmcl.2018.01.022DOI Listing
April 2018

Identification of Staphylococcus aureus Cellular Pathways Affected by the Stilbenoid Lead Drug SK-03-92 Using a Microarray.

Antibiotics (Basel) 2017 Sep 11;6(3). Epub 2017 Sep 11.

Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53211, USA.

The mechanism of action for a new lead stilbene compound coded SK-03-92 with bactericidal activity against methicillin-resistant (MRSA) is unknown. To gain insight into the killing process, transcriptional profiling was performed on SK-03-92 treated vs. untreated . Fourteen genes were upregulated and 38 genes downregulated by SK-03-92 treatment. Genes involved in sortase A production, protein metabolism, and transcriptional regulation were upregulated, whereas genes encoding transporters, purine synthesis proteins, and a putative two-component system (SACOL2360 (MW2284) and SACOL2361 (MW2285)) were downregulated by SK-03-92 treatment. Quantitative real-time polymerase chain reaction analyses validated upregulation of and as well as downregulation of the MW2284/MW2285 and purine biosynthesis genes in the drug-treated population. A quantitative real-time polymerase chain reaction analysis of and mutants compared to wild-type cells demonstrated that the gene was upregulated by both putative two-component regulatory gene mutants compared to the wild-type strain. Using a transcription profiling technique, we have identified several cellular pathways regulated by SK-03-92 treatment, including a putative two-component system that may regulate and other genes that could be tied to the SK-03-92 mechanism of action, biofilm formation, and drug persisters.
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http://dx.doi.org/10.3390/antibiotics6030017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617981PMC
September 2017

A Small-Molecule Inhibitor of Iron-Sulfur Cluster Assembly Uncovers a Link between Virulence Regulation and Metabolism in Staphylococcus aureus.

Cell Chem Biol 2016 Nov 20;23(11):1351-1361. Epub 2016 Oct 20.

Department of Pathology, Microbiology, & Immunology, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Veterans Affairs Tennessee Valley Healthcare Services, Nashville, TN 37232, USA. Electronic address:

The rising problem of antimicrobial resistance in Staphylococcus aureus necessitates the discovery of novel therapeutic targets for small-molecule intervention. A major obstacle of drug discovery is identifying the target of molecules selected from high-throughput phenotypic assays. Here, we show that the toxicity of a small molecule termed '882 is dependent on the constitutive activity of the S. aureus virulence regulator SaeRS, uncovering a link between virulence factor production and energy generation. A series of genetic, physiological, and biochemical analyses reveal that '882 inhibits iron-sulfur (Fe-S) cluster assembly most likely through inhibition of the Suf complex, which synthesizes Fe-S clusters. In support of this, '882 supplementation results in decreased activity of the Fe-S cluster-dependent enzyme aconitase. Further information regarding the effects of '882 has deepened our understanding of virulence regulation and demonstrates the potential for small-molecule modulation of Fe-S cluster assembly in S. aureus and other pathogens.
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http://dx.doi.org/10.1016/j.chembiol.2016.09.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5117899PMC
November 2016

Zinc Pyrithione Improves the Antibacterial Activity of Silver Sulfadiazine Ointment.

mSphere 2016 Sep-Oct;1(5). Epub 2016 Sep 14.

Department of Ophthalmology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.

Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus are commonly associated with biofilm-associated wound infections that are recalcitrant to conventional antibiotics. As an initial means to identify agents that may have a greater propensity to improve clearance of wound-associated bacterial pathogens, we screened a Food and Drug Administration-approved drug library for members that display bactericidal activity toward 72-h-established P. aeruginosa biofilms using an adenylate kinase reporter assay for bacterial cell death. A total of 34 compounds displayed antibiofilm activity. Among these, zinc pyrithione was also shown to reduce levels of A. baumannii and S. aureus biofilm-associated bacteria and exhibited an additive effect in combination with silver sulfadiazine, a leading topical therapeutic for wound site infections. The improved antimicrobial activity of zinc pyrithione and silver sulfadiazine was maintained in an ointment formulation and led to improved clearance of P. aeruginosa, A. baumannii, and S. aureus in a murine model of wound infection. Taken together, these results suggest that topical zinc pyrithione and silver sulfadiazine combination formulations may mitigate wound-associated bacterial infections and disease progression. IMPORTANCE Topical antimicrobial ointments ostensibly mitigate bacterial wound disease and reliance on systemic antibiotics. Yet studies have called into question the therapeutic benefits of several traditional topical antibacterials, accentuating the need for improved next-generation antimicrobial ointments. Yet the development of such agents consisting of a new chemical entity is a time-consuming and expensive proposition. Considering that drug combinations are a mainstay therapeutic strategy for the treatment of other therapeutic indications, one alternative approach is to improve the performance of conventional antimicrobial ointments by the addition of a well-characterized and FDA-approved agent. Here we report data that indicate that the antimicrobial properties of silver sulfadiazine ointments can be significantly improved by the addition of the antifungal zinc pyrithione, suggesting that such combinations may provide an improved therapeutic option for the topical treatment of wound infections.
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http://dx.doi.org/10.1128/mSphere.00194-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5023846PMC
September 2016

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection.

PLoS Pathog 2015 Dec 18;11(12):e1005341. Epub 2015 Dec 18.

Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America.

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.
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http://dx.doi.org/10.1371/journal.ppat.1005341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684308PMC
December 2015

Neomycin Sulfate Improves the Antimicrobial Activity of Mupirocin-Based Antibacterial Ointments.

Antimicrob Agents Chemother 2016 Feb 23;60(2):862-72. Epub 2015 Nov 23.

Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA

In the midst of the current antimicrobial pipeline void, alternative approaches are needed to reduce the incidence of infection and decrease reliance on last-resort antibiotics for the therapeutic intervention of bacterial pathogens. In that regard, mupirocin ointment-based decolonization and wound maintenance practices have proven effective in reducing Staphylococcus aureus transmission and mitigating invasive disease. However, the emergence of mupirocin-resistant strains has compromised the agent's efficacy, necessitating new strategies for the prevention of staphylococcal infections. Herein, we set out to improve the performance of mupirocin-based ointments. A screen of a Food and Drug Administration (FDA)-approved drug library revealed that the antibiotic neomycin sulfate potentiates the antimicrobial activity of mupirocin, whereas other library antibiotics did not. Preliminary mechanism of action studies indicate that neomycin's potentiating activity may be mediated by inhibition of the organism's RNase P function, an enzyme that is believed to participate in the tRNA processing pathway immediately upstream of the primary target of mupirocin. The improved antimicrobial activity of neomycin and mupirocin was maintained in ointment formulations and reduced S. aureus bacterial burden in murine models of nasal colonization and wound site infections. Combination therapy improved upon the effects of either agent alone and was effective in the treatment of contemporary methicillin-susceptible, methicillin-resistant, and high-level mupirocin-resistant S. aureus strains. From these perspectives, combination mupirocin-and-neomycin ointments appear to be superior to that of mupirocin alone and warrant further development.
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http://dx.doi.org/10.1128/AAC.02083-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4750660PMC
February 2016

Clinically Relevant Growth Conditions Alter Acinetobacter baumannii Antibiotic Susceptibility and Promote Identification of Novel Antibacterial Agents.

PLoS One 2015 11;10(11):e0143033. Epub 2015 Nov 11.

Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America.

Biological processes that govern bacterial proliferation and survival in the host-environment(s) are likely to be vastly different from those that are required for viability in nutrient-rich laboratory media. Consequently, growth-based antimicrobial screens performed in conditions modeling aspects of bacterial disease states have the potential to identify new classes of antimicrobials that would be missed by screens performed in conventional laboratory media. Accordingly, we performed screens of the Selleck library of 853 FDA approved drugs for agents that exhibit antimicrobial activity toward the Gram-negative bacterial pathogen Acinetobacter baumannii during growth in human serum, lung surfactant, and/or the organism in the biofilm state and compared those results to that of conventional laboratory medium. Results revealed that a total of 90 compounds representing 73 antibiotics and 17 agents that were developed for alternative therapeutic indications displayed antimicrobial properties toward the test strain in at least one screening condition. Of the active library antibiotics only four agents, rifampin, rifaximin, ciprofloxacin and tetracycline, exhibited antimicrobial activity toward the organism during all screening conditions, whereas the remainder were inactive in ≥ 1 condition; 56 antibiotics were inactive during serum growth, 25 and 38 were inactive toward lung surfactant grown and biofilm-associated cells, respectively, suggesting that subsets of antibiotics may outperform others in differing infection settings. Moreover, 9 antibiotics that are predominantly used for the treatment Gram-positive pathogens and 10 non-antibiotics lacked detectable antimicrobial activity toward A. baumannii grown in conventional medium but were active during ≥ 1 alternative growth condition(s). Such agents may represent promising anti-Acinetobacter agents that would have likely been overlooked by antimicrobial whole cell screening assays performed in traditional laboratory screening media.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143033PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4641712PMC
June 2016

Editorial overview: Antimicrobials.

Curr Opin Microbiol 2015 Oct 15;27:v-vii. Epub 2015 Sep 15.

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http://dx.doi.org/10.1016/j.mib.2015.09.001DOI Listing
October 2015

Translational deficiencies in antibacterial discovery and new screening paradigms.

Curr Opin Microbiol 2015 Oct 8;27:108-13. Epub 2015 Sep 8.

BacterioScan, Inc., St. Louis, MO 63108, United States. Electronic address:

An impending disaster is currently developing in the infectious disease community: the combination of rapidly emerging multidrug-resistance among clinically relevant bacterial pathogens, together with an unprecedented withdrawal from industrial dedication to this disease area, is jeopardizing human health on a societal level. For those who remain focused and dedicated to identifying solutions to this growing problem, additional challenges await when in vitro activity does not correlate with in vivo efficacy. Thus the development of more effective translational assays will greatly improve and streamline the process of identifying novel antibacterial agents that can stand the test of preclinical and clinical development. Here we describe recent examples of research that justify the need for such assays.
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http://dx.doi.org/10.1016/j.mib.2015.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659729PMC
October 2015

In the midst of the antimicrobial discovery conundrum: an overview.

Curr Opin Microbiol 2015 Oct 8;27:103-7. Epub 2015 Sep 8.

Department of Microbiology and Immunology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642, United States. Electronic address:

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http://dx.doi.org/10.1016/j.mib.2015.08.005DOI Listing
October 2015

A LysR-family transcriptional regulator required for virulence in Brucella abortus is highly conserved among the α-proteobacteria.

Mol Microbiol 2015 Oct 14;98(2):318-28. Epub 2015 Aug 14.

Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24060, USA.

Small RNAs are principal elements of bacterial gene regulation and physiology. Two small RNAs in Brucella abortus, AbcR1 and AbcR2, are required for wild-type virulence. Examination of the abcR loci revealed the presence of a gene encoding a LysR-type transcriptional regulator flanking abcR2 on chromosome 1. Deletion of this lysR gene (bab1_1517) resulted in the complete loss of abcR2 expression while no difference in abcR1 expression was observed. The B. abortus bab1_1517 mutant strain was significantly attenuated in macrophages and mice, and bab1_1517 was subsequently named vtlR for virulence-associated transcriptional LysR-family regulator. Microarray analysis revealed three additional genes encoding small hypothetical proteins also under the control of VtlR. Electrophoretic mobility shift assays demonstrated that VtlR binds directly to the promoter regions of abcR2 and the three hypothetical protein-encoding genes, and DNase I footprint analysis identified the specific nucleotide sequence in these promoters that VtlR binds to and drives gene expression. Strikingly, orthologs of VtlR are encoded in a wide range of host-associated α-proteobacteria, and it is likely that the VtlR genetic system represents a common regulatory circuit critical for host-bacterium interactions.
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http://dx.doi.org/10.1111/mmi.13123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846693PMC
October 2015

A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

PLoS One 2015 22;10(6):e0129234. Epub 2015 Jun 22.

Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America; Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.

Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0129234PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476654PMC
March 2016

Rot is a key regulator of Staphylococcus aureus biofilm formation.

Mol Microbiol 2015 Apr 26;96(2):388-404. Epub 2015 Feb 26.

Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA, 52242, USA.

Staphylococcus aureus is a significant cause of chronic biofilm infections on medical implants. We investigated the biofilm regulatory cascade and discovered that the repressor of toxins (Rot) is part of this pathway. A USA300 community-associated methicillin-resistant S. aureus strain deficient in Rot was unable to form a biofilm using multiple different assays, and we found rot mutants in other strain lineages were also biofilm deficient. By performing a global analysis of transcripts and protein production controlled by Rot, we observed that all the secreted protease genes were up-regulated in a rot mutant, and we hypothesized that this regulation could be responsible for the biofilm phenotype. To investigate this question, we determined that Rot bound to the protease promoters, and we observed that activity levels of these enzymes, in particular the cysteine proteases, were increased in a rot mutant. By inactivating these proteases, biofilm capacity was restored to the mutant, demonstrating they are responsible for the biofilm negative phenotype. Finally, we tested the rot mutant in a mouse catheter model of biofilm infection and observed a significant reduction in biofilm burden. Thus S. aureus uses the transcription factor Rot to repress secreted protease levels in order to build a biofilm.
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http://dx.doi.org/10.1111/mmi.12943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467170PMC
April 2015
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