Structure-based discovery of a small-molecule inhibitor of methicillin-resistant Staphylococcus aureus virulence

The rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains poses a major threat to public health. MRSA possesses an arsenal of secreted host-damaging virulence factors that mediate pathogenicity and blunt immune defenses. Panton–Valentine leukocidin (PVL) and α-toxin are exotoxins that create lytic pores in the host cell membrane. They are recognized as being important for the development of invasive MRSA infections and are thus potential targets for antivirulence therapies. Here, we report the high-resolution X-ray crystal structures of both PVL and α-toxin in their soluble, monomeric, and oligomeric membrane-inserted pore states in complex with n-tetradecylphosphocholine (C14PC). The structures revealed two evolutionarily conserved phosphatidylcholine-binding mechanisms and their roles in modulating host cell attachment, oligomer assembly, and membrane perforation. Moreover, we demonstrate that the soluble C14PC compound protects primary human immune cells in vitro against cytolysis by PVL and α-toxin and hence may serve as the basis for the development of an antivirulence agent for managing MRSA infections.

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Structure-Based Discovery of a Novel Small-Molecule Inhibitor of Methicillin-Resistant S. aureus

10.1101/781971 ◽

2019 ◽

Author(s):

Jie Liu ◽

Lina Kozhaya ◽

Victor J. Torres ◽

Derya Unutmaz ◽

Min Lu

Keyword(s):

Cell Attachment ◽

Immune Defense ◽

Methicillin Resistant ◽

Molecule Inhibitor ◽

Membrane Perforation ◽

Host Cell Attachment ◽

Mrsa Infection ◽

Mrsa Strains ◽

Antivirulence Agents

SummaryThe rapid emergence and dissemination of methicillin-resistant Staphylococcus aureus (MRSA) strains represents a major threat to public health. MRSA elaborates an arsenal of secreted host-damaging virulence factors to mediate pathogenicity and blunt immune defense. Panton-Valentine leukocidin (PVL) and α-toxin are pore-forming cytotoxins of recognized importance in the development of invasive MRSA infection and are thus potential targets for antivirulence therapy. We report the X-ray crystal structures of PVL and α-toxin in their soluble, monomeric and oligomeric, membrane-inserted pore states, in complex with n-tetradecylphosphocholine (C14PC). The structures reveal two evolutionarily conserved phosphatidylcholine binding mechanisms and their roles in modulating host cell attachment, oligomer assembly and membrane perforation. Moreover, we demonstrate that the soluble C14PC compound protects primary human immune cells in vitro against cytolysis by PVL and α-toxin and hence may serve as the basis for the development of novel antivirulence agents to combat MRSA.

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Evaluation of Ceftaroline Alone and in Combination against Biofilm-Producing Methicillin-Resistant Staphylococcus aureus with Reduced Susceptibility to Daptomycin and Vancomycin in anIn VitroPharmacokinetic/Pharmacodynamic Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00386-15 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4497-4503 ◽

Cited By ~ 23

Author(s):

Katie E. Barber ◽

Jordan R. Smith ◽

Cortney E. Ireland ◽

Blaise R. Boles ◽

Warren E. Rose ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Medical Device ◽

Bloodstream Infections ◽

Methicillin Resistant ◽

Content Type ◽

Elimination Half ◽

Antimicrobial Protection ◽

Mrsa Strains ◽

Post Hoc

ABSTRACTAnnually, medical device infections are associated with >250,000 catheter-associated bloodstream infections (CLABSI), with up to 25% mortality.Staphylococcus aureus, a primary pathogen in these infections, is capable of biofilm production, allowing organism persistence in harsh environments, offering antimicrobial protection. With increases inS. aureusisolates with reduced susceptibility to current agents, ceftaroline (CPT) offers a therapeutic alternative. Therefore, we evaluated whether CPT would have a role against biofilm-producing methicillin-resistantS. aureus(MRSA), including those with decreased susceptibilities to alternative agents. In this study, we investigated CPT activity alone or combined with daptomycin (DAP) or rifampin (RIF) against 3 clinical biofilm-producing MRSA strains in anin vitrobiofilm pharmacokinetic/pharmacodynamic (PK/PD) model. Simulated antimicrobial regimens were as follows: 600 mg of CPT every 8 h (q8h) (free maximum concentration of drug [fCmax], 17.04 mg/liter; elimination half-life [t1/2], 2.66 h), 12 mg/kg of body weight/day of DAP (fCmax, 14.7 mg/liter;t1/2, 8 h), and 450 mg of RIF q12h (fCmax, 3.5 mg/liter;t1/2, 3.4 h), CPT plus DAP, and CPT plus RIF. Samples were obtained and plated to determine colony counts. Differences in log10CFU/cm2were evaluated by analysis of variance with Tukey'spost hoctest. The strains were CPT and vancomycin susceptible and DAP nonsusceptible (DNS). CPT displayed activity throughout the experiment. DAP demonstrated initial activity with regrowth at 24 h in all strains. RIF was comparable to the drug-free control, and little benefit was observed when combined with CPT. CPT plus DAP displayed potent activity, with an average log10CFU/cm2reduction of 3.33 ± 1.01 from baseline. CPT demonstrated activity against biofilm-producing DNS MRSA. CPT plus DAP displayed therapeutic enhancement over monotherapy, providing a potential option for difficult-to-treat medical device infections.

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Clinical Relevance and Antimicrobial Profiling of Methicillin-Resistant Staphylococcus aureus (MRSA) on Routine Antibiotics and Ethanol Extract of Mango Kernel (Mangifera indica L.)

BioMed Research International ◽

10.1155/2020/4150678 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8

Author(s):

Ali Al Bshabshe ◽

Martin R. P. Joseph ◽

Amgad A. Awad El-Gied ◽

Abdalla N. Fadul ◽

Harish C. Chandramoorthy ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Clinical Relevance ◽

Mangifera Indica ◽

Diffusion Method ◽

Drug Candidate ◽

Methicillin Resistant ◽

Mango Kernel ◽

Ethanolic Extracts ◽

Mrsa Strains

Methicillin-resistant Staphylococcus aureus (MRSA) is known for serious health problems. Testing new inexpensive natural products such as mango kernel (Mangifera indica L., Anacardiaceae) may provide alternative and economically viable anti-MRSA drugs. In the current study, we screened clinical isolates from Aseer Central Hospital, Saudi Arabia, during 2012–2017 for MRSA and tested an ethanolic extract of mango kernel for anti-MRSA activity. Brief confirmation of MRSA was performed by the Vitek 2 system, while antibiotic sensitivity of strains was tested for their clinical relevance. The In vitro disc diffusion method was used to test the anti-MRSA activity of the ethanolic mango kernel extract. The antimicrobial activity of mango kernel was compared to that of standard drugs (oxacillin and vancomycin). Of the identified 132 S. aureus strains, 42 (31.8%) were found to be MRSA and their prevalence showed a clear increase during the last two years (2016-2017; p<0.001). MRSA strains showed 100% sensitivity to vancomycin, teicoplanin, linezolid, tetracycline, daptomycin, tigecycline, and tobramycin and 100% resistance to ampicillin and 98% to penicillin. The ethanolic extracts of mango kernel were found active against both S. aureus and the MRSA strains. Inhibitory activities (mean ± SE) were achieved at concentrations of 50 mg/mL (20.77 ± 0.61), 5 mg/mL (16.18 ± 0.34), and 0.5 mg/mL (8.39 ± 0.33) exceeding that of vancomycin (p=0.0162). MRSA strains were sensitive to mango kernel extracts when compared to vancomycin. Therefore, ethanolic extracts of mango kernel can be escalated to animal model studies as a promising leading anti-MRSA drug candidate and can be an economic alternative to high-priced synthetic antibiotics.

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A structure-based rationale for sialic acid independent host-cell entry of Sosuga virus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906717116 ◽

2019 ◽

Vol 116 (43) ◽

pp. 21514-21520 ◽

Cited By ~ 3

Author(s):

Alice J. Stelfox ◽

Thomas A. Bowden

Keyword(s):

Sialic Acid ◽

Host Cell ◽

Receptor Binding ◽

Cell Attachment ◽

Cell Entry ◽

Head Region ◽

Close Proximity ◽

Host Cell Attachment ◽

Host Cell Entry

The bat-borne paramyxovirus, Sosuga virus (SosV), is one of many paramyxoviruses recently identified and classified within the newly established genus Pararubulavirus, family Paramyxoviridae. The envelope surface of SosV presents a receptor-binding protein (RBP), SosV-RBP, which facilitates host-cell attachment and entry. Unlike closely related hemagglutinin neuraminidase RBPs from other genera of the Paramyxoviridae, SosV-RBP and other pararubulavirus RBPs lack many of the stringently conserved residues required for sialic acid recognition and hydrolysis. We determined the crystal structure of the globular head region of SosV-RBP, revealing that while the glycoprotein presents a classical paramyxoviral six-bladed β-propeller fold and structurally classifies in close proximity to paramyxoviral RBPs with hemagglutinin-neuraminidase (HN) functionality, it presents a receptor-binding face incongruent with sialic acid recognition. Hemadsorption and neuraminidase activity analysis confirms the limited capacity of SosV-RBP to interact with sialic acid in vitro and indicates that SosV-RBP undergoes a nonclassical route of host-cell entry. The close overall structural conservation of SosV-RBP with other classical HN RBPs supports a model by which pararubulaviruses only recently diverged from sialic acid binding functionality.

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Complementation of the fadA Mutation in Fusobacterium nucleatum Demonstrates that the Surface-Exposed Adhesin Promotes Cellular Invasion and Placental Colonization

Infection and Immunity ◽

10.1128/iai.00209-09 ◽

2009 ◽

Vol 77 (7) ◽

pp. 3075-3079 ◽

Cited By ~ 48

Author(s):

Akihiko Ikegami ◽

Peter Chung ◽

Yiping W. Han

Keyword(s):

Host Cell ◽

Cell Attachment ◽

Adverse Pregnancy Outcome ◽

Fusobacterium Nucleatum ◽

Host Cells ◽

Mouse Placenta ◽

Host Cell Attachment ◽

Adverse Pregnancy

ABSTRACT Fusobacterium nucleatum is a gram-negative oral anaerobe implicated in periodontal disease and adverse pregnancy outcome. The organism colonizes the mouse placenta, causing localized infection and inflammation. The mechanism of placental colonization has not been elucidated. Previous studies identified a novel adhesin from F. nucleatum, FadA, as being involved in the attachment and invasion of host cells. The fadA deletion mutant F. nucleatum 12230 US1 was defective in host cell attachment and invasion in vitro, but it also exhibited pleiotropic effects with altered cell morphology and growth rate. In this study, a fadA-complementing clone, F. nucleatum 12230 USF81, was constructed. The expression of FadA on USF81 was confirmed by Western blotting and immunofluorescent labeling. USF81 restored host cell attachment and invasion activities. The ability of F. nucleatum 12230, US1, and USF81 to colonize the mouse placenta was examined. US1 was severely defective in placental colonization compared to the wild type and USF81. Thus, FadA plays an important role in F. nucleatum colonization in vivo. These results also represent the first complementation studies for F. nucleatum. FadA may be a therapeutic target for preventing F. nucleatum colonization of the host.

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A bacteriocin-based antimicrobial formulation to effectively disrupt the cell viability of methicillin-resistant Staphylococcus aureus (MRSA) biofilms

npj Biofilms and Microbiomes ◽

10.1038/s41522-020-00166-4 ◽

2020 ◽

Vol 6 (1) ◽

Author(s):

Christian Kranjec ◽

Kirill V. Ovchinnikov ◽

Torstein Grønseth ◽

Kumar Ebineshan ◽

Aparna Srikantam ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Viability ◽

Cell Damage ◽

Penicillin G ◽

Antibiotic Resistant ◽

Methicillin Resistant ◽

Lactam Antibiotic ◽

Mrsa Strains ◽

Therapeutic Tools

AbstractAntibiotic-resistant and biofilm-associated infections brought about by methicillin-resistant Staphylococcus aureus (MRSA) strains is a pressing issue both inside as well as outside nosocomial environments worldwide. Here, we show that a combination of two bacteriocins with distinct structural and functional characteristics, garvicin KS, and micrococcin P1, showed a synergetic antibacterial activity against biofilms produced in vitro by S. aureus, including several MRSA strains. In addition, this bacteriocin-based antimicrobial combination showed the ability to restore the sensitivity of the highly resilient MRSA strain ATCC 33591 to the β-lactam antibiotic penicillin G. By using a combination of bacterial cell metabolic assays, confocal and scanning electron microscopy, we show that the combination between garvicin KS, micrococcin P1, and penicillin G potently inhibit cell viability within S. aureus biofilms by causing severe cell damage. Together these data indicate that bacteriocins can be valuable therapeutic tools in the fight against biofilm-associated MRSA infections.

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Ceftobiprole EfficacyIn Vitroagainst Panton-Valentine Leukocidin Production andIn Vivoagainst Community-Associated Methicillin-Resistant Staphylococcus aureus Osteomyelitis in Rabbits

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00926-12 ◽

2012 ◽

Vol 56 (12) ◽

pp. 6291-6297 ◽

Cited By ~ 9

Author(s):

Azzam Saleh-Mghir ◽

Oana Dumitrescu ◽

Aurélien Dinh ◽

Yassine Boutrad ◽

Laurent Massias ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant ◽

Content Type ◽

The Mean ◽

Mrsa Strains ◽

Time Kill ◽

Panton Valentine Leukocidin ◽

Valentine Leukocidin

ABSTRACTCommunity-associated methicillin-resistantStaphylococcus aureus(CA-MRSA) can cause osteomyelitis with severe sepsis and/or local complications in which a Panton-Valentine leukocidin (PVL) role is suspected.In vitrosub-MIC antibiotic effects on growth and PVL production by 11 PVL+MRSA strains, including the major CA-MRSA clones (USA300, including the LAC strain; USA400; and USA1000), and 11 PVL+methicillin-susceptibleS. aureus(MSSA) strains were tested in microplate culture. Time-kill analyses with ceftobiprole at its MIC were also run with LAC. Efficacies of ceftobiprole (40 mg/kg of body weight subcutaneously [s.c.] four times a day [q.i.d.]) or vancomycin (60 mg/kg intramuscularly [i.m.] twice a day [b.i.d.]) alone or combined with rifampin (10 mg/kg b.i.d.) against rabbit CA-MRSA osteomyelitis, induced by tibial injection of 3.4 × 107CFU of LAC, were compared. Treatment, started 14 days postinoculation, lasted 14 days.In vitro, 6/11 strains cultured with sub-MICs of ceftobiprole produced 1.6- to 4.8-fold more PVL than did the controls, with no link to specific clones. Rifampin decreased PVL production by all tested strains. In time-kill analyses at the LAC MIC (0.75 mg/liter), PVL production rose transiently at 6 and 8 h and then declined 2-fold at 16 h, concomitant with a 2-log10-CFU-count decrease.In vivo, the mean log10CFU/g of bone for ceftobiprole (1.44 ± 0.40) was significantly lower than that for vancomycin (2.37 ± 1.22) (P= 0.034), with 7/10 versus 5/11 bones sterilized, respectively. Combination with rifampin enhanced ceftobiprole (1.16 ± 0.04 CFU/g of bone [P= 0.056], 11/11 sterile bones) and vancomycin (1.23 ± 0.06 CFU/g [P= 0.011], 11/11 sterile bones) efficacies. Ceftobiprole bactericidal activity and the rifampin anti-PVL effect could play a role in these findings, which should be of interest for treating CA-MRSA osteomyelitis.

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Comparative Efficacies of Human Simulated Exposures of Telavancin and Vancomycin against Methicillin-Resistant Staphylococcus aureus with a Range of Vancomycin MICs in a Murine Pneumonia Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00062-10 ◽

2010 ◽

Vol 54 (12) ◽

pp. 5115-5119 ◽

Cited By ~ 36

Author(s):

Jared L. Crandon ◽

Joseph L. Kuti ◽

David P. Nicolau

Keyword(s):

Staphylococcus Aureus ◽

Bacterial Load ◽

Infection Model ◽

Time Curve ◽

Unbound Fraction ◽

Methicillin Resistant ◽

Vancomycin Mics ◽

Mrsa Strains

ABSTRACT Telavancin displays potent in vitro and in vivo activity against methicillin-resistant Staphylococcus aureus (MRSA), including strains with reduced susceptibility to vancomycin. We compared the efficacies of telavancin and vancomycin against MRSA strains with vancomycin MICs of ≥1 μg/ml in a neutropenic murine lung infection model. Thirteen clinical MRSA isolates (7 vancomycin-susceptible, 2 vancomycin-heteroresistant [hVISA], and 4 vancomycin-intermediate [VISA] isolates) were tested after 24 h, and 7 isolates (1 hVISA and 4 VISA isolates) were tested after 48 h of exposure. Mice were administered subcutaneous doses of telavancin at 40 mg/kg of body weight every 12 h (q12h) or of vancomycin at 110 mg/kg q12h; doses were designed to simulate the area under the concentration-time curve for the free, unbound fraction of drug (fAUC) observed for humans given telavancin at 10 mg/kg q24h or vancomycin at 1 g q12h. Efficacy was expressed as the 24- or 48-h change in lung bacterial density from pretreatment counts. At dose initiation, the mean bacterial load was 6.16 ± 0.26 log10 CFU/ml, which increased by averages of 1.26 ± 0.55 and 1.74 ± 0.68 log in untreated mice after 24 and 48 h, respectively. At both time points, similar CFU reductions were noted for telavancin and vancomycin against MRSA, with vancomycin MICs of ≤2 μg/ml. Both drugs were similarly efficacious after 24 and 48 h of treatment against the hVISA strains tested. Against VISA isolates, telavancin reduced bacterial burdens significantly more than vancomycin for 1 of 4 isolates after 24 h and for 3 of 4 isolates after 48 h. These data support the potential utility of telavancin for the treatment of MRSA pneumonia caused by pathogens with reduced susceptibility to vancomycin.

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Missense Mutations in PBP2A Affecting Ceftaroline Susceptibility Detected in Epidemic Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Clonotypes ST228 and ST247 in Western Switzerland Archived since 1998

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04068-14 ◽

2015 ◽

Vol 59 (4) ◽

pp. 1922-1930 ◽

Cited By ~ 52

Author(s):

William L. Kelley ◽

Ambre Jousselin ◽

Christine Barras ◽

Emmanuelle Lelong ◽

Adriana Renzoni

Keyword(s):

Staphylococcus Aureus ◽

University Hospital ◽

Surveillance Program ◽

Missense Mutations ◽

Unknown Parameters ◽

Methicillin Resistant ◽

Content Type ◽

Resistant Strains ◽

Mrsa Strains

ABSTRACTThe development and maintenance of an arsenal of antibiotics is a major health care challenge. Ceftaroline is a new cephalosporin with activity against methicillin-resistantStaphylococcus aureus(MRSA); however, no reports concerning MRSA ceftaroline susceptibility have been reported in Switzerland. We tested thein vitroactivity of ceftaroline against an archived set of 60 MRSA strains from the University Hospital of Geneva collected from 1994 to 2003. Our results surprisingly revealed ceftaroline-resistant strains (MIC, >1 μg/ml in 40/60 strains; EUCAST breakpoints, susceptible [S], ≤1 μg/ml; resistant [R], >1 μg/ml) were present from 1998 to 2003. The detected resistant strains predominantly belonged to sequence type 228 (ST228) (South German clonotype) but also to ST247 (Iberian clonotype). A sequence analysis of these strains revealed missense mutations in the penicillin-binding protein 2A (PBP2A) allosteric domain (N146K or E239K and N146K-E150K-G246E). The majority of our ST228 PBP2A mutations (N146K or E150K) were distinct from ST228 PBP2A allosteric domain mutations (primarily E239K) recently described for MRSA strains collected in Thailand and Spain during the 2010 Assessing Worldwide Antimicrobial Resistance Evaluation (AWARE) global surveillance program. We also found that similar allosteric domain PBP2A mutations (N146K) correlated with ceftaroline resistance in an independent external ST228 MRSA set obtained from the nearby University Hospital of Lausanne, Lausanne, Switzerland, collected from 2003 to 2008. Thus, ceftaroline resistance was observed in our archived strains (including two examples of an MIC of 4 µg/ml for the Iberian ST247 clonotype with the triple mutation N146K/E150K/G246E), at least as far back as 1998, considerably predating the commercial introduction of ceftaroline. Our results reinforce the notion that unknown parameters can potentially exert selective pressure on PBP2A that can subsequently modulate ceftaroline resistance.

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Antimicrobial Constituents from Machaerium Pers.: Inhibitory Activities and Synergism of Machaeriols and Machaeridiols against Methicillin-Resistant Staphylococcus aureus, Vancomycin-Resistant Enterococcus faecium, and Permeabilized Gram-Negative Pathogens

Molecules ◽

10.3390/molecules25246000 ◽

2020 ◽

Vol 25 (24) ◽

pp. 6000

Author(s):

Ilias Muhammad ◽

Melissa R. Jacob ◽

Mohamed A. Ibrahim ◽

Vijayasankar Raman ◽

Mallika Kumarihamy ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Enterococcus Faecium ◽

Methicillin Resistant Staphylococcus Aureus ◽

Polymyxin B ◽

Synergistic Activity ◽

Vancomycin Resistant Enterococcus ◽

Methicillin Resistant ◽

Vancomycin Resistant ◽

Mrsa Strains

Two new epimeric bibenzylated monoterpenes machaerifurogerol (1a) and 5-epi-machaerifurogerol (1b), and four known isoflavonoids (+)-vestitol (2), 7-O-methylvestitol (3), (+)-medicarpin (4), and 3,8-dihydroxy-9-methoxypterocarpan (5) were isolated from Machaerium Pers. This plant was previously assigned as Machaerium multiflorum Spruce, from which machaeriols A-D (6–9) and machaeridiols A-C (10–12) were reported, and all were then re-isolated, except the minor compound 9, for a comprehensive antimicrobial activity evaluation. Structures of the isolated compounds were determined by full NMR and mass spectroscopic data. Among the isolated compounds, the mixture 10 + 11 was the most active with an MIC value of 1.25 μg/mL against methicillin-resistant Staphylococcus aureus (MRSA) strains BAA 1696, −1708, −1717, −33591, and vancomycin-resistant Enterococcus faecium (VRE 700221) and E. faecalis (VRE 51299) and vancomycin-sensitive E. faecalis (VSE 29212). Compounds 6–8 and 10–12 were found to be more potent against MRSA 1708, and 6, 11, and 12 against VRE 700221, than the drug control ciprofloxacin and vancomycin. A combination study using an in vitro Checkerboard method was carried out for machaeriols (7 or 8) and machaeridiols (11 or 12), which exhibited a strong synergistic activity of 12 + 8 (MIC 0.156 and 0.625 µg/mL), with >32- and >8-fold reduction of MIC’s, compared to 12, against MRSA 1708 and −1717, respectively. In the presence of sub-inhibitory concentrations on polymyxin B nonapeptide (PMBN), compounds 10 + 11, 11, 12, and 8 showed activity in the range of 0.5–8 µg/mL for two strains of Acinetobacter baumannii, 2–16 µg/mL against Pseudomonas aeruginosa PAO1, and 2 µg/mL against Escherichia coli NCTC 12923, but were inactive (MIC > 64 µg/mL) against the two isolates of Klebsiella pneumoniae.

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