Fluorescent Bocillins: Synthesis and application in the detection of penicillin-binding proteins

Gee, Kyle R.; Kang, Hee Young; Meier, Timothy I.; Zhao, Genshi; Blaszcak, Larry C.

doi:10.1002/1522-2683()22:5<960::aid-elps960>3.0.co;2-9

Cited by 37 publications

(21 citation statements)

References 19 publications

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“…A total of 1.33 g/l of membrane proteins was labeled with 25 M BOCILLIN-FL (Molecular Probes, Eugene, OR) in 50 mM sodium phosphate buffer, pH 7.0, for 15 min at 37°C (18,63). Afterward, proteins were denatured at 95°C in 1ϫ sample buffer.…”

Section: Methodsmentioning

confidence: 99%

Characterization of Osmotically Induced Filaments of Salmonella enterica

Pratt

Chen

Czuprynski

et al. 2012

Appl Environ Microbiol

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ABSTRACTSalmonella entericaforms aseptate filaments with multiple nucleoids when cultured in hyperosmotic conditions. These osmotic-induced filaments are viable and form single colonies on agar plates even though they contain multiple genomes and have the potential to divide into multiple daughter cells. Introducing filaments that are formed during osmotic stress into culture conditions without additional humectants results in the formation of septa and their division into individual cells, which could present challenges to retrospective analyses of infectious dose and risk assessments. We sought to characterize the underlying mechanisms of osmotic-induced filament formation. The concentration of proteins and chromosomal DNA in filaments and control cells was similar when standardized by biomass. Furthermore, penicillin-binding proteins in the membrane of salmonellae were activein vitro. The activity of penicillin-binding protein 2 was greater in filaments than in control cells, suggesting that it may have a role in osmotic-induced filament formation. Filaments contained more ATP than did control cells in standardized cell suspensions, though the levels of two F0F1-ATP synthase subunits were reduced. Furthermore, filaments could septate and divide within 8 h in 0.2× Luria-Bertani broth at 23°C, while nonfilamentous control cells did not replicate. Based upon the ability of filaments to septate and divide in this diluted broth, a method was developed to enumerate by plate count the number of individual, viable cells within a population of filaments. This method could aid in retrospective analyses of infectious dose of filamented salmonellae.

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Section: Methodsmentioning

confidence: 99%

Characterization of Osmotically Induced Filaments of Salmonella enterica

Pratt

Chen

Czuprynski

et al. 2012

Appl Environ Microbiol

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“…The comparators, chosen to represent narrow-spectrum, expanded-spectrum, and broad-spectrum cephalosporins (cephalexin, cefuroxime, and ceftriaxone, respectively) and cephamycins (cefoxitin), were obtained from Sigma-Aldrich, St. Louis, MO, or Teva Pharma, Wilrijk, Belgium. Gentamicin, used to control extracellular growth in the absence of another antibiotic (7), was obtained as Geomycin (distributed in Belgium by Glaxo-SmithKline SA, Genval, Belgium); Bocillin FL (a fluorescent derivative of penicillin V [13]) was obtained from Invitrogen Corp., Carlsbad, CA. Staphylococcal PBP 2a (soluble form) was obtained from Escherichia coli Rosetta 2(DE3) transformed with plasmid pET28a carrying the truncated PBP 2a-coding sequence (⌬M1-Y23 PBP 2a).…”

Section: Methodsmentioning

confidence: 99%

“…The PBP 2a binding of ceftobiprole and the other cephalosporins tested was assessed by countermarking experiments by following the general method described for 3 H-labeled penicillin G (29, 37) but by using Bocillin FL (a boron-dipyrromethene [bodipy] derivative of penicillin V [13]) as the reporter antibiotic. The purified PBP 2a was incubated at 37°C for 25 min with increasing concentrations of the cephalosporins under study, after which 100 M Bocillin FL was added to the samples for an additional 25 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Activities of Ceftobiprole and Other Cephalosporins against Extracellular and Intracellular (THP-1 Macrophages and Keratinocytes) Forms of Methicillin-Susceptible and Methicillin-ResistantStaphylococcus aureus

Lemaire

Glupczynski

Duval

et al. 2009

Antimicrob Agents Chemother

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Restricted to the hospital setting for many years, the methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) epidemic is now reaching an increasing variety of other environments (12), such as patients in the community in various parts of the world (16,35,41) and animals (21, 40). Beyond its spectacular ability to adapt and to develop resistance to most antimicrobial agents (9), including drugs of last resort, such as vancomycin, linezolid, and daptomycin (5, 28, 31), the capacity of S. aureus to invade, sojourn, and thrive intracellularly (8, 23, 34) creates an additional challenge since intracellular forms tend to be poorly susceptible to most available antibiotics (38). Evaluations of new antistaphylococcal agents directed against resistant S. aureus strains must therefore include an assessment of their ability to control intracellular infections. While animal models of staphylococcal infection are being developed (32), models of cultured cells remain useful because they offer the possibility to explore in detail the pharmacological parameters governing the response of the intracellular bacteria to the drug in the absence of host factors (7,38). In this context, we observed that, contrary to most original assumptions (36), the poor accumulation of ␤-lactams in phagocytic cells does not preclude the observation of significant activity against intracellular methicillin-susceptible S. aureus (MSSA). This is actually dependent on the time of exposure (12 to 24 h) and whether the extracellular concentration is maintained at a sufficiently large but still clinically pertinent level (7,20). We previously reported that intraphagocytic MRSA isolates regain almost full susceptibility to penicillins and carbapenems, due to the acidic pH prevailing in phagolysosomes (19). This finding has been rationalized by the observation that acidic pH improves the accessibility to and the acylation of PBP 2a by penicillins within a time frame relevant to the growth rate of MRSA through protein conformational changes (17). This triggered us to study ceftobiprole in this context. Ceftobiprole, also known as BAL9141 and Ro 63-9141 (4), is the first clinically developed cephalosporin that shows almost similar activities against MRSA and MSSA isolates in conventional in vitro tests (15,42). It has now been approved for clinical use in some countries and has been studied in a large array of preclinical and clinical settings (see references 3 and 43 for recent reviews).

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“…b-glucuronidase and total protein, as well as penicillinbinding proteins and total protein have also been described recently [25,26]. The cited methods involve detecting the specialized subclass of proteins using an application-specific green fluorophore, followed by staining of total protein with SYPRO Ruby protein gel stain.…”

Section: Stain Compatibility With Dichromatic Detection Methodsmentioning

confidence: 99%

An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

Berggren¹,

Schulenberg²,

Lopez³

et al. 2002

Proteomics

133

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SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix-assisted laser desorption/ionisation-time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post-staining gels subjected to labeling with other fluorophores such as Schiff's base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post-staining gels after detection of glycoproteins using the green fluorescent Pro-Q Emerald 300 glycoprotein stain or detection of beta-glucuronidase using the green fluorescent ELF 97 beta-D-glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.

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Fluorescent Bocillins: Synthesis and application in the detection of penicillin-binding proteins

Cited by 37 publications

References 19 publications

Characterization of Osmotically Induced Filaments of Salmonella enterica

Characterization of Osmotically Induced Filaments of Salmonella enterica

Activities of Ceftobiprole and Other Cephalosporins against Extracellular and Intracellular (THP-1 Macrophages and Keratinocytes) Forms of Methicillin-Susceptible and Methicillin-ResistantStaphylococcus aureus

An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

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