Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe

Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis ( Mtb ), however, makes this challenging at the point of care, particularly in resource-limited settings....

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Vydané v:	Nature chemistry Ročník 4; číslo 10; s. 802 - 809
Hlavní autori:	Xie, Hexin, Mire, Joseph, Kong, Ying, Chang, MiHee, Hassounah, Hany A., Thornton, Chris N., Sacchettini, James C., Cirillo, Jeffrey D., Rao, Jianghong
Médium:	Journal Article
Jazyk:	English
Vydavateľské údaje:	London Nature Publishing Group UK 01.10.2012 Nature Publishing Group
Predmet:	639/638/11 639/638/45 Analytical Chemistry Bacterial Proteins - metabolism beta-Lactamases - metabolism Binding Sites Biochemistry Catalytic Domain Chemistry Chemistry and Materials Science Chemistry/Food Science Computer Simulation Crystallization Emissions Enzymes Fluorescence Fluorescent Dyes - chemistry Fluorescent Dyes - metabolism Humans Hydrolysis Inorganic Chemistry Kinetics Mortality Mycobacterium tuberculosis Mycobacterium tuberculosis - enzymology Mycobacterium tuberculosis - isolation & purification Organic Chemistry Pathogens Physical Chemistry Point-of-Care Systems Probes Protein Structure, Tertiary Spectrometry, Fluorescence Sputum - microbiology Substrate Specificity Tuberculosis Tuberculosis - diagnosis United States > US Texas
ISSN:	1755-4330, 1755-4349, 1755-4349
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Abstract	Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis ( Mtb ), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100–200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe ( CDG-OMe ) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens. Rapid diagnostic methods that can be applied in resource-limited settings are important in the fight against tuberculosis. Here, fluorogenic probes are described that are activated by BlaC — an enzyme secreted by tubercle bacilli. The probes have enabled detection in unprocessed human sputum of live pathogen in less than 10 min.
AbstractList	Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100-200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens. Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis ( Mtb ), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100–200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe ( CDG-OMe ) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens. Rapid diagnostic methods that can be applied in resource-limited settings are important in the fight against tuberculosis. Here, fluorogenic probes are described that are activated by BlaC — an enzyme secreted by tubercle bacilli. The probes have enabled detection in unprocessed human sputum of live pathogen in less than 10 min. Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100-200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 [beta]-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens. Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100-200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens.Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative pathogen, Mycobacterium tuberculosis (Mtb), however, makes this challenging at the point of care, particularly in resource-limited settings. Here we report the use of BlaC (an enzyme naturally expressed/secreted by tubercle bacilli) as a marker and the design of BlaC-specific fluorogenic substrates as probes for Mtb detection. These probes showed an enhancement by 100-200 times in fluorescence emission on BlaC activation and a greater than 1,000-fold selectivity for BlaC over TEM-1 β-lactamase, an important factor in reducing false-positive diagnoses. Insight into the BlaC specificity was revealed by successful co-crystallization of the probe/enzyme mutant complex. A refined green fluorescent probe (CDG-OMe) enabled the successful detection of live pathogen in less than ten minutes, even in unprocessed human sputum. This system offers the opportunity for the rapid, accurate detection of very low numbers of Mtb for the clinical diagnosis of tuberculosis in sputum and other specimens.
Author	Mire, Joseph Xie, Hexin Rao, Jianghong Sacchettini, James C. Chang, MiHee Cirillo, Jeffrey D. Thornton, Chris N. Kong, Ying Hassounah, Hany A.
AuthorAffiliation	3 Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, Bryan, Texas 77807, USA 2 Departments of Chemistry and Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA 4 Global BioDiagnostics Corp., 19 N. Main St, Temple, Texas 76501, USA 1 Molecular Imaging Program at Stanford, Departments of Radiology and Chemistry, Stanford University, 1201 Welch Road, Stanford, California 94305-5484, USA
AuthorAffiliation_xml	– name: 4 Global BioDiagnostics Corp., 19 N. Main St, Temple, Texas 76501, USA – name: 2 Departments of Chemistry and Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843, USA – name: 3 Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center, Bryan, Texas 77807, USA – name: 1 Molecular Imaging Program at Stanford, Departments of Radiology and Chemistry, Stanford University, 1201 Welch Road, Stanford, California 94305-5484, USA
Author_xml	– sequence: 1 givenname: Hexin surname: Xie fullname: Xie, Hexin organization: Departments of Radiology and Chemistry, Molecular Imaging Program at Stanford, Stanford University – sequence: 2 givenname: Joseph surname: Mire fullname: Mire, Joseph organization: Departments of Chemistry and Biochemistry and Biophysics, Texas A&M University – sequence: 3 givenname: Ying surname: Kong fullname: Kong, Ying organization: Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center – sequence: 4 givenname: MiHee surname: Chang fullname: Chang, MiHee organization: Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center – sequence: 5 givenname: Hany A. surname: Hassounah fullname: Hassounah, Hany A. organization: Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center – sequence: 6 givenname: Chris N. surname: Thornton fullname: Thornton, Chris N. organization: Global BioDiagnostics Corp – sequence: 7 givenname: James C. surname: Sacchettini fullname: Sacchettini, James C. organization: Departments of Chemistry and Biochemistry and Biophysics, Texas A&M University – sequence: 8 givenname: Jeffrey D. surname: Cirillo fullname: Cirillo, Jeffrey D. organization: Department of Microbial and Molecular Pathogenesis, Texas A&M Health Science Center – sequence: 9 givenname: Jianghong surname: Rao fullname: Rao, Jianghong email: jrao@stanford.edu organization: Departments of Radiology and Chemistry, Molecular Imaging Program at Stanford, Stanford University
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Snippet	Early diagnosis of tuberculosis can dramatically reduce both its transmission and the associated death rate. The extremely slow growth rate of the causative...
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SubjectTerms	639/638/11 639/638/45 Analytical Chemistry Bacterial Proteins - metabolism beta-Lactamases - metabolism Binding Sites Biochemistry Catalytic Domain Chemistry Chemistry and Materials Science Chemistry/Food Science Computer Simulation Crystallization Emissions Enzymes Fluorescence Fluorescent Dyes - chemistry Fluorescent Dyes - metabolism Humans Hydrolysis Inorganic Chemistry Kinetics Mortality Mycobacterium tuberculosis Mycobacterium tuberculosis - enzymology Mycobacterium tuberculosis - isolation & purification Organic Chemistry Pathogens Physical Chemistry Point-of-Care Systems Probes Protein Structure, Tertiary Spectrometry, Fluorescence Sputum - microbiology Substrate Specificity Tuberculosis Tuberculosis - diagnosis
Title	Rapid point-of-care detection of the tuberculosis pathogen using a BlaC-specific fluorogenic probe
URI	https://link.springer.com/article/10.1038/nchem.1435 https://www.ncbi.nlm.nih.gov/pubmed/23000993 https://www.proquest.com/docview/1069248987 https://www.proquest.com/docview/1074766021 https://www.proquest.com/docview/1125230078 https://pubmed.ncbi.nlm.nih.gov/PMC4136554
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